KR102717836B1 - 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 improving autoimmune diseases, comprising as an active ingredient a compound represented by chemical formula 1 isolated from a perilla leaf extract and a pharmaceutically acceptable salt thereof.

Description

{Composition for preventing or treating autoimmune disease comprising compound isolated from Paeonia lactiflora extract as effective component}

The present invention 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, to a composition comprising a compound isolated from a perilla leaf extract as an active ingredient, and which can be used for preventing, improving or treating autoimmune diseases by inhibiting differentiation into Th17 cells and production of IL-17.

CD4 ⁺ T cells can be divided into subtypes such as Th1 (T helper type 1), Th2 (T helper type 2), CD4 ⁺ CD25 ⁺ regulatory T cells, and Th17 cells depending on the expression of specific cytokines and transcription factors. Among these, Th17 cells that produce IL-17 have been identified as having a direct role in inflammation and autoimmune pathogenesis, as well as in host defense against pathogens or inducing abnormal immune responses through animal model and human cell studies. As the major pathogenic roles of IL-17 and Th17 cells in inflammation and autoimmune diseases have been identified, their suppression and regulation have been suggested as important strategies for disease treatment, and potential targeted treatment methods through suppression and neutralization of Th17 cell lineage cytokines and suppression and regulation of specific transcription factors are currently being studied.

Rheumatoid arthritis is an autoimmune disease that affects various tissues and organs of the body. Its symptoms include severe pain in joints such as the joints of the fingers, and causes abnormalities in various organs such as muscles, lungs, heart, skin, blood vessels, nerves, and eyes in addition to the joints. Even if the above symptoms do not appear significantly, they gradually progress over time and develop 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. Symptoms include frequent abdominal pain and diarrhea, and in severe cases, fever and vaginal bleeding.

It has been reported that one of the causes of autoimmune diseases such as rheumatoid arthritis and Crohn's disease is the increased expression of IL-17 due to differentiation of TH17. In fact, IL-17 has been observed to enhance granulocyte formation (granulopoiesis) by promoting the expression of G-CSF and GM-CSF, and to promote germinal center formation and autoantibody production. In relation to this, IL-17 is detected at high levels in the serum of patients with rheumatoid arthritis, an inflammatory autoimmune disease, and IL-17A induces the expression of IL-1β and IL-6 in the synovial cells of patients with rheumatoid arthritis. In addition, it is known that the main function of IL-17 is to inhibit matrix production in chondrocytes and osteoblasts, causing joint damage and leading to deficiency of tissue regeneration.

Many patients suffer from autoimmune diseases such as rheumatoid arthritis and Crohn's disease because there is currently no treatment or preventive treatment available. Therefore, there is an urgent need to develop preventive and therapeutic agents for autoimmune diseases that are effective, safe, and do not cause side effects.

The researchers of the present invention, while developing a preventive and therapeutic agent for autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease, and Crohn's disease, discovered that a perilla leaf extract and tormentic acid, a compound isolated therefrom, can be effectively applied to such diseases, thereby completing the present invention.

Korean Patent No. 10-1970457

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

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

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

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 a compound represented by the following chemical formula 1 or a salt thereof separated from a perilla leaf extract as an active ingredient:

[Chemical Formula 1]

.

(2) A pharmaceutical composition for preventing or treating an autoimmune disease, wherein the extraction solvent of the above-mentioned perilla leaf extract is water, a C ₁ to C ₄ alcohol, or a mixture thereof.

(3) A pharmaceutical composition for preventing or treating an autoimmune disease, wherein the compound is separated by adding at least one solvent selected from the group consisting of water, C ₁ to C ₅ straight-chain or branched alcohol, hexane, dichloromethane, and ethyl acetate to a perilla leaf extract.

(4) A pharmaceutical composition for preventing or treating an autoimmune disease, wherein the compound is separated by adding at least one solvent selected from the group consisting of hexane, dichloromethane, ethyl acetate, butanol, and water to a perilla leaf extract.

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

(6) A pharmaceutical composition for preventing or treating an autoimmune disease, wherein the separation is performed by fractionating the dichloromethane layer obtained by fractionating with dichloromethane using silica gel column chromatography.

(7) A pharmaceutical composition for preventing or treating autoimmune diseases, which is separated using the gradient method of solvent concentration by silica gel column chromatography.

(8) A pharmaceutical composition for preventing or treating an autoimmune disease, wherein the salt is a physiologically acceptable salt with an inorganic acid, an organic acid, an inorganic base or an organic base.

(9) A pharmaceutical composition for preventing or treating an autoimmune disease, 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 at least one selected from the group consisting of citric acid, acetic acid, lactic acid, tartariac 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, glutamic acid, and aspartic acid.

(10) A pharmaceutical composition for preventing or treating an autoimmune disease, wherein the pharmaceutical composition further comprises a diluent or carrier.

(11) A pharmaceutical composition for preventing or treating autoimmune diseases, formulated as an oral preparation or a parenteral preparation.

(12) A pharmaceutical composition for preventing or treating an autoimmune disease, wherein the oral preparation is a tablet, capsule, pill, powder, granule, suspension or syrup, and the parenteral preparation is a cream, lotion, ointment, solution, gel, cataplasma, patch, aerosol, fluid extract, elixir, infusion, sachet or injection.

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

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

A method for producing a pharmaceutical composition for preventing or treating an autoimmune disease, comprising: a step of obtaining a compound of chemical formula 1 of claim 1 by separating the dichloromethane fraction obtained from the above step using silica gel column chromatography;

(14) A food composition for preventing or treating autoimmune diseases, comprising a compound represented by the following chemical formula 1 or a salt thereof separated from a perilla leaf extract as an effective ingredient:

[Chemical Formula 1]

.

The compound represented by chemical formula 1 or a salt thereof isolated from a perilla leaf extract according to one aspect can effectively inhibit the production of IL-17, and more specifically, can effectively inhibit the expression of IL-17 by inhibiting the polarization of Th17 cells. Accordingly, a composition containing the compound or a salt thereof as an effective ingredient can be used for the prevention, improvement, or treatment of autoimmune diseases.

Figure 1 shows the solvent fractionation process (scheme) of the perilla leaf extract.
Figure 2A shows the cell viability (%) according to the toxicity evaluation of each ethanol extract of perilla leaves, Cheonnyeoncho fruit, Cheonnyeoncho stem, and Chrysanthemum indicum, and Figure 2B shows the IL-17A expression rate (%) of each ethanol extract of perilla leaves, Cheonnyeoncho fruit, Cheonnyeoncho stem, Chrysanthemum indicum, and green tea.
Figure 3A shows the cell viability (%) according to the toxicity evaluation of the perilla leaf fraction, and Figure 3B shows the IL-17A expression rate (%) of the perilla leaf fraction.
Figure 4A shows the cell viability (%) according to the toxicity evaluation of the dichloromethane (DCM) fraction of the perilla leaves, and Figure 4B shows the IL-17A expression rate (%) of the DCM fraction.
Figure 5 shows the IL-17A expression rate (%) of each subfraction of dichloromethane (DCM) from the leaves.
Figure 6 shows the TLC results of the PFD4-2 subfraction.
Figure 7A shows the ¹ H-NMR spectrum (400 MHz, CD ₃ OD) results of PFD4-2-5-6.
Figure 7B shows the ¹³ C-NMR spectrum (100 MHz, CD ₃ OD) results of PFD4-2-5-6.
Figure 7C shows the structure of tomentic acid, a compound isolated as an active ingredient from the dichloromethane fraction of perilla leaves.
Figure 8 shows the separation process of tomentic acid from the dichloromethane fraction of the ethanol extract of perilla leaves.
Figure 9 shows the IL-17A expression rate (%) measured using PFD 4-2-5-6 (tormentic acid) and rosmarinic acid isolated from the perilla leaves.
Figure 10 shows the IL-17A expression rate (%) measured using ethanol extract of perilla leaves, PFD 4-2-5-6 (tormentic acid) isolated from perilla leaves, tomentic acid standard, and PFD 2-3-2-2 (ursolic acid).
Figure 11 shows the treatment process of the DSS-induced inflammatory enteritis model with the extract of perilla leaves and tomentic acid isolated therefrom.
Figures 12A and 12B show the results of measuring the change in intestinal length after treatment with perilla leaf extract, tomentic acid, and mesalazine in an animal model of DSS-induced inflammatory enteritis.
Figures 13A and 13B show histological changes in the intestine in an animal model of DSS-induced inflammatory enteritis.
Figures 14A, 14B, and 14C show the CD4 ⁺ IL-17 ⁺ cell expression rate (%) in the 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, are used with the same meaning as commonly understood by those skilled in the art in the relevant field of the present invention. In addition, although preferred methods or samples are described in this specification, similar or equivalent ones are also included in the scope of the present invention. The contents of all publications mentioned as references in this specification are incorporated herein by reference in their entirety.

One aspect provides a pharmaceutical composition for preventing or treating an autoimmune disease, comprising a compound represented by the following chemical formula 1 or a salt thereof isolated from a perilla leaf extract as an active ingredient.

[Chemical Formula 1]

The term "autoimmune disease" as used herein refers to a disease in which the immune system abnormally attacks its own cells or causes an abnormal reaction in its own organs. Examples thereof include rheumatoid arthritis, Crohn's disease, progressive systemic sclerosis (Scleroderma), systemic lupus erythematosus (lupus), insulin-dependent juvenile diabetes mellitus due to pancreatic cell antibodies, multiple sclerosis, autoimmune hemolytic anemia, myasthenia gravis, Grave's disease, or inflammatory bowel disease.

In one specific example, 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 mucosa.

The term "extraction" used in this specification means separating the components of a herbal medicine in order to incorporate them into a pharmaceutical composition. However, depending on the specific details of each step, it may be interpreted as meaning the first step of separating an effective ingredient from a herbal medicine.

In this specification, "Perilla leaves ( Perilla frutescens var. acuta )" refers to the leaves of Perilla frutescens var. acuta KUDO., an annual herb belonging to the Lamiaceae family.

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

The alcohol may be from 1 to 100% (v/v). Alternatively, the alcohol may be provided by diluting the alcohol in water, for example, by diluting the alcohol in water from 1 to 99% (v/v).

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

According to one specific example, ethanol is a preferred extraction solvent for extracting high content of tomentic acid from the perilla leaf extract. For example, since water extraction hardly contains tomentic acid, ethanol extraction is more preferred. In addition, since methanol extraction has toxicity due to methanol, ethanol extraction is more preferred for use as a pharmaceutical composition or food composition.

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 the perilla leaves. The "volume times" above means the volume of the solvent compared to the weight of the perilla leaves, i.e., a multiple corresponding to ml per g.

The extraction temperature of the above extract may be performed at 4°C to 120°C. In addition, the extraction time may be 12 hours to 120 hours. If the extraction temperature is lower than 4°C, extraction may not occur properly, and if it exceeds 120°C, the extract may be denatured. If the extraction time is lower than 12 hours, some of the effective ingredient may not be extracted, and if it exceeds 120 hours, impurity components other than the effective ingredient may be extracted together, which is not preferable. Alternatively, the extraction temperature may be room temperature.

Depending on the requirements, the extraction process can be repeated 1 to 10 times, for example 2 to 5 times.

The above-mentioned perilla leaf extract may be manufactured according to a manufacturing method of a conventional plant extract, and specifically, may be manufactured by a cold extraction method, a warm extraction method, a heat extraction method, an ultrasonic extraction method, etc., and may utilize a conventional extraction device, an ultrasonic grinding extractor, or a fractionator.

The above-mentioned perilla leaf extract can be stored at room temperature after extraction with an extraction solvent, removal of perilla leaf residue, filtering with filter paper, and concentrating the filtrate using a vacuum rotary concentrator or vacuum dryer.

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

When two or more types of fractionating solvents are used, the fractions may be individual solvent fractions obtained by performing solvent fractionation using two or more types of fractionating solvents simultaneously or sequentially. For example, the fractions may be individual solvent fractions 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 specific example, the compound of the chemical formula 1 can be obtained as a fraction by further fractionating the perilla leaf extract using water or one or more organic solvents selected from the group consisting of hexane, dichloromethane, ethyl acetate, butanol, and chloroform, simultaneously or sequentially.

In one specific example, the compound is isolated by adding one or more solvents selected from the group consisting of water, C ₁ to C ₅ straight-chain or branched alcohols, hexane, dichloromethane, and ethyl acetate to a perilla leaf extract.

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

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

The fractionation temperature may be performed at 4°C to 120°C. 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 a product dried or concentrated by a known method as described above.

Depending on the requirements, the fractionation process can be repeated 1 to 10 times, for example 2 to 5 times.

If necessary, the extract may be suspended prior to fractionation.

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

In one specific example, the separation can be performed by fractionating with dichloromethane and separating the obtained dichloromethane layer using silica gel column chromatography.

In one specific example, silica gel column chromatography can separate using a solvent concentration gradient method.

In one specific example, the salt can be a physiologically acceptable salt with an inorganic acid, an organic acid, an inorganic base or an organic base.

In one specific example, the inorganic acid may be at least one selected from the group consisting of hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid, and the organic acid may be at least one selected from the group consisting of citric acid, acetic acid, lactic acid, tartariac 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, glutamic acid, and aspartic acid.

The above salt may be a pharmaceutically acceptable salt or a food-based acceptable salt. The term "pharmaceutically acceptable salt" or "food-based acceptable salt" generally refers to a salt that is safe, non-toxic, and not biologically or otherwise unsuitable and thus can be used in the preparation of pharmaceutical and food-based compositions.

The compound of the above chemical formula 1 can be synthesized in vitro or extracted from a herb. When extracted from a herb, it can be separated and extracted from the perilla leaves.

The compound of the above chemical formula 1 may be referred to as tormentic acid.

The tomentose compound represented by the above chemical formula 1 or a salt thereof can be involved in regulating cell differentiation and reduce the expression of IL-17A, a Th17-related cytokine and transcription factor.

Th17 cells are an essential T cell subset that accumulates at mucosal surfaces and protects the host from bacterial and fungal infections. In addition to their protective immune functions, they also serve as important effectors of autoimmune inflammation. Th17 cells are characterized by the secretion of the cytokines IL-17A, IL-17F, IL-21, and IL-22. Unregulated or inappropriate activation of Th17 cells plays a key role in several inflammatory pathologies.

The tomentate compound represented by the above chemical formula 1 or a salt thereof can effectively inhibit the production of IL-17, and more specifically, inhibit the polarization of Th17, thereby inhibiting the expression of IL-17. Accordingly, a composition containing the above compound or a salt thereof as an effective ingredient can be used for preventing, improving, or treating autoimmune diseases.

The term “T helper 17 cells (Th17, Th17 cells, T helper 17 cells)” as used herein refers to a population of pro-inflammatory helper T cells that produce interleukin 17 (IL-17). They are related to regulatory T cells and are associated with signals that differentiate Th17s and inhibit actual regulatory T cell differentiation. Th17s are developmentally distinct from Th1 and Th2. Th17 cells play an important role in maintaining the mucosal barrier and contribute to the elimination of pathogens from mucosal surfaces, but are also associated with autoimmune diseases and inflammatory diseases. A decrease in Th17 cells at mucosal surfaces is also associated with chronic inflammation and microbial transfer.

A compound represented by chemical formula 1 or a pharmaceutically acceptable salt thereof isolated from a perilla leaf extract provides excellent use in preventing, improving or treating autoimmune diseases.

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

The above derivative refers to a compound obtained by replacing part of the structure of the above compound with another atom or atomic group. The term "substitution" refers to the introduction in place of a hydrogen atom when forming a derivative by replacing one or more hydrogen atoms in an organic compound with another atomic group, and "substituent" refers to the introduced atomic group. The substituent can be, for example, a hydroxy group, a halogen atom (e.g., 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, a C ₁ to C ₁₀ heteroalkyl group, a C ₆ to C ₁₀ aryl group, a C ₆ to C ₁₀ heteroaryl group, a C ₁ to C ₁₀ alkoxy, a nitro group, a cyano group, an amino group, a carboxyl group or a salt thereof, a sulfonyl group, a sulfamoyl group, a sulfonic acid group or a salt thereof, phosphoric acid or a salt thereof, or a combination thereof.

The above isomers may be structural isomers or stereoisomers.

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

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

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

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

In one specific example, the oral preparation may be exemplified as a tablet, a capsule, a pill, a powder, a liquid, a dry syrup, a granule, a suspension, or a syrup, but is not limited thereto. The parenteral preparation may be exemplified as a cream, a lotion, an ointment, a solution, a gel, a cataplasma, a patch, an aerosol, a fluid extract, an elixir, a sachet, an injection, a skin emulsifier, a skin suspension, a transdermal patch, a drug-containing bandage, but is not limited thereto.

The composition may be administered by any method known in the art. For example, it may 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 systemic or local.

Another aspect is a step of extracting the perilla leaves with 50% (v/v) to 100% (v/v) ethanol;

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

The present invention provides a method for producing a pharmaceutical composition for preventing or treating an autoimmune disease, comprising: a step of obtaining a compound of the chemical formula 1 by separating the dichloromethane fraction obtained from the above step using silica gel column chromatography;

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

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

The above food composition can also be provided as a dietary supplement or health functional food.

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

The term "prevention" as used herein means inhibiting the occurrence of a disease or condition in a subject who has not been diagnosed as having the disease or condition, but is predisposed to developing such disease or condition.

The term "treatment" as used herein means (a) inhibition of the development of a disease or condition, (b) alleviation of the disease or condition, and (c) elimination of the disease or condition in a subject.

The term "improvement" as used herein means any act that improves the symptoms of a disease or condition in a subject.

The term "subject" as used herein refers to a mammal, such as a monkey, cow, horse, pig, sheep, dog, cat, rat, mouse, chimpanzee, or the like, including a human, having a disease whose symptoms can be improved by administering the composition of the present invention.

The term "including as an active ingredient" as used herein means including a sufficient amount to achieve the efficacy or activity of a compound represented by Chemical Formula 1, a derivative thereof, or a mixture thereof separated from a perilla leaf extract. For example, the content of the active ingredient in a pharmaceutical composition for preventing or treating an autoimmune disease may be 0.001 wt% to 99.9 wt%, 0.1 wt% to 99 wt%, or 1 wt% to 50 wt%, but is not limited thereto, and may be appropriately adjusted to a desired content depending on the usage form and usage method of the composition.

In one specific example, the compound represented by the chemical formula 1, a derivative thereof, or a mixture thereof isolated from a perilla leaf extract may be included in an amount of 1 to 80 wt% based on the total weight of the pharmaceutical composition, and may be provided in the form of a solvate or a pharmaceutically acceptable salt.

In one specific example, the compound represented by the chemical formula 1, a derivative thereof, or a mixture thereof isolated from a perilla leaf extract may be included in an amount of 1 to 80 wt% based on the total weight of the food composition, and may be provided in the form of a solvate or a food-chemically acceptable salt.

In addition, the composition may additionally include a known autoimmune disease treatment agent in addition to the above-mentioned effective ingredient.

The compound represented by Chemical Formula 1, a derivative thereof, or a mixture thereof in the composition may be included in a pharmaceutically or food-wise effective amount. For example, the compound represented by Chemical Formula 1, a derivative thereof, or a mixture thereof may be present in the composition at a concentration of 0.001 to 1000 nM. For example, the compound represented by Chemical Formula 1, a derivative thereof, or a mixture thereof may be present in the composition at a concentration of 5 nM, 10 nM, 25 nM, 30 nM, 40 nM, 50 nM, 60 nM, 75 nM, 100 nM, or a range where these values are the upper or lower limits. For example, the compound may be present in the composition at a concentration of 1 to 10 M.

Alternatively, the compound represented by chemical formula 1 in the composition, a derivative thereof, or a mixture thereof may be present in a range of 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 where these are the upper or lower limits, for example, 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 chemical formula 1, a derivative thereof, or a mixture thereof may be included in the composition in an amount of 0.1 μg/mL or more.

For example, the compound represented by chemical formula 1, a derivative thereof, or a mixture thereof in the composition may be included in an amount equal to all, 1/2, or 1/3 of the daily dosage required for preventing, treating, or improving an autoimmune disease.

Alternatively, the concentration of the ethanol extract of perilla leaves or the dichloromethane fraction separated therefrom in the composition may be 0.001 to 100 μg/mL, for example, 0.5, 1, 2, 4, 5, 7, 8, 10, 15, 20, 30 μg/mL or a range where these are the upper or lower limits, for example, 0.5 to 30 μg/mL, 1 to 20 μg/mL, 4 to 15 μg/mL, 7 to 10 μg/mL.

Alternatively, the composition may contain an ethanol extract of the perilla leaves or a dichloromethane fraction separated therefrom in an amount of 0.1 ㎍/mL or more.

The above composition can be prepared in unit dose form by formulating it using pharmaceutically or food-wise acceptable additives or carriers, or can be prepared by placing it in a multi-dose container.

The appropriate dosage of the composition varies depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity, and a skilled physician can easily determine and prescribe an effective dosage for the desired treatment, prevention, or improvement. According to one specific example, the daily dosage of the composition may be 0.001 to 10 g/kg.

For example, the dosage of a pharmaceutical composition for treating an autoimmune disease can be determined by considering the absorbability and inactivation rate of the active ingredient and the drug to be used concomitantly, and can be administered at 0.1 mg/kg (body weight) 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) of the active ingredient per day, and can be administered once or in several divided doses, but is not limited thereto.

For example, the ethanol extract of perilla leaves, the dichloromethane fraction thereof, or the compound represented by formula 1 isolated therefrom, a derivative thereof, or a mixture thereof can be administered at 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 intended to exemplify the present invention and the scope of the present invention is not limited to these examples.

Example 1. Preparation of ethanol extract of perilla leaves

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

Example 2. Preparation of fractions from ethanol extract of perilla leaves

The ethanol extract of perilla leaves prepared in Example 1 was fractionated into immiscible solvents by utilizing 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 three times, and the concentrate was stored at 4°C until use.

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

More specifically, Figure 1 shows the solvent fractionation process of the ethanol extract of perilla leaves using n-hexane, dichloromethane (DCM), ethyl acetate (EtOAc), butanol, and water.

Comparative Example 1. Preparation of ethanol extracts of Thousand-year-old berry, Thousand-year-old vine stem, Chrysanthemum indicum, and green tea

Ethanol extracts from the fruit of Cheonnyeoncho, Cheonnyeoncho stem, Chrysanthemum indicum, and green tea were obtained using the same method as the method for producing the ethanol extract of the perilla leaves described in Example 1.

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

The ethanol extract of the perilla leaves obtained in Example 1 and the ethanol extracts of the fruits of the common ginseng, the stem of the common ginseng, the Chinese chives, and the green tea obtained in Comparative Example 1 were used to evaluate toxicity and measure the expression level of IL-17A.

(1) Toxicity assessment (measurement of cell activity)

After extracting the pancreas from 6-week-old B6 mice, CD4 ⁺ T cells were separated using MACS positive separation using CD4-beads, and 5 x 10 ⁵ cells were seeded in a 96-multiwell plate and cultured in a 5% CO ₂ incubator. The cultured cells were treated with ethanol extracts (2, 5, and 10 ㎍/mL) of perilla leaves, Cheonnyeoncho fruit, Cheonnyeoncho stem, and Chrysanthemum indicum, respectively, and then cultured for 1 day. After MTT was added, the cells were cultured at 37°C for 4 hours. After that, 100 ul of 0.04 N HCl in isopropanol was added to dissolve the formazan, and the cell activity was measured by measuring the absorbance at 570 nm using a microplate reader (Emax, Molecular Devices). The DMSO treatment group was used as the control group.

Figure 2A shows the cell viability (%) according to the toxicity evaluation of each ethanol extract of the leaves of the plant, the fruit of the plant, the stem of the plant, and the root of the plant.

More specifically, Fig. 2A shows the results of the toxicity evaluation measured at different concentrations of 2, 5, and 10 ㎍/mL for each ethanol extract.

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

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

After extracting the pancreas from 6-week-old B6 mice, CD4+ T cells were separated using CD4-beads by MACS positive separation. The separated CD4 ⁺ T cells were seeded at 1.2 x 10 ⁶ cells on 12-multiwell plates coated with anti-CD3 and CD28 (each 1 μg/ml) antibodies, 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 ethanol extracts of perilla leaves, Cheonnyeoncho fruit, Cheonnyeoncho stem, Chrysanthemum indicum, and green tea (5 and 10 μg/mL), and then cultured for 1-3 days. After staining with CD4-APC and IL-17A-PE intracellular staining kits, the IL-17A expression level was measured by FACS. The DMSO treatment group was used as the control group.

Figure 2B shows the IL-17A expression rate (%) of each ethanol extract of perilla leaves, Angelica dahurica fruit, Angelica dahurica stem, Chrysanthemum indicum, and green tea.

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

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

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

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

(1) Toxicity assessment (measurement of cell activity)

After removing the pancreas from 6-week-old B6 mice, CD4 ⁺ T cells were separated using MACS positive separation using CD4-beads, and 5 x 10 ⁵ cells were seeded in a 96-multiwell plate and cultured in a 5% CO ₂ incubator. The cultured cells were treated with hexane, dichloromethane, ethynacetate, butanol, and water fractions of the ethanol extract of perilla leaves at concentrations of 10, 20, 50, and 100 ㎍/mL, respectively, and then cultured for 1 day. After MTT was added, the cells were cultured at 37°C for 4 hours. After that, 100 ul of 0.04 N HCl in isopropanol was added to dissolve the formazan, and the cell activity was measured by measuring the absorbance at 570 nm using a microplate reader (Emax, Molecular Devices). The DMSO treatment group was used as the control group.

Figure 3A shows the cell viability (%) according to the toxicity evaluation of the hexane fraction (HEX FR.), dichloromethane fraction (DCM FR.), ethyl acetate fraction (EA FR.), butanol fraction (BuOH FR.), and water fraction (Water FR.) of the perilla leaves.

More specifically, Figure 3A shows the results of the toxicity evaluation measured at different concentrations of each of the above leaf fractions: 10, 20, 50, and 100 ㎍/mL.

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

After extracting the pancreas from 6-week-old B6 mice, CD4+ T cells were separated using CD4-beads by MACS positive separation. The separated CD4 ⁺ T cells were seeded at 1.2 x 10 ⁶ cells on 12-multiwell plates coated with anti-CD3 and CD28 (each 1 μg/ml) antibodies, 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, ethynacetate, butanol, and water fractions of the ethanol extract of perilla leaves at concentrations of 10, 20, 50, and 100 μg/mL, respectively, and then cultured for 1-3 days. After staining with CD4-APC and IL-17A-PE intracellular staining kits, the IL-17A expression level was measured by FACS. The DMSO treatment group was used as the control group.

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

More specifically, Fig. 3B shows the IL-17A expression rate (%) measured at different concentrations of each of the leaflet fractions, 10, 20, 50, and 100 ㎍/mL.

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

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

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

(1) Toxicity assessment (measurement of cell activity)

The dichloromethane fraction obtained from the ethanol extract of the perilla leaves was treated to cultured cells at concentrations of 1, 5, and 10 ㎍/mL, and the toxicity was measured using the same method as in Test Example 2.

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

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

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

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

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

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

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

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

As shown in Figure 4B, all dichloromethane fractions of the perilla leaves effectively inhibited IL-17A expression at concentrations of 0.1 μg/mL or higher.

Example 3. Separation of dichloromethane subfraction of perilla leaves

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

Figure 5 shows the IL-17A expression rate (%) of each subfraction of dichloromethane (DCM) from the leaves.

As a result, fraction 4 (PFD4), which had a generally high IL-17 expression inhibitory activity, was selected.

Example 4. Isolation of active ingredient from dichloromethane fraction of perilla leaves

(1) Isolation of active ingredients from the DCM fraction of the perilla leaves

The 4th fraction (PFD4) of the dichloromethane fraction of perilla leaves obtained by the method of Example 3 was selected and the effective ingredient was continuously separated.

PFD4 was obtained from among the six subfractions obtained by applying it to open column chromatography (chloroform : methanol = 100 : 1 ~ 20 : 1 ~ 1 : 1 ~ 0 : 1) using silica gel as a stationary phase. Among these, the PFD4-2 layer with the largest amount was selected and subjected to Silica Open Column Chromatography (chloroform : methanol = 100 : 1 ~ 0 : 1) to obtain seven subfractions again. Among these, the PFD4-2-5 layer (232 mg) was selected and subjected to Silica Open Column Chromatography (chloroform : acetone = 4.5 : 1 ~ 3 : 1 ~ 1 : 1 ~ 0 : 1, methanol 100%), and as a result, a single substance of fraction 6 (4-2-5-6) was secured as an active ingredient.

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

(2) Identification of compounds of active ingredients separated from the DCM fraction of the perilla leaves

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

Figure 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 those in the literature, and the compound structure was identified as tormentic acid.

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).

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

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

Test Example 4. Evaluation of Th17 Inhibitory Effect

The Th17 inhibitory effect was measured using PFD 4-2-5-6 (tormentic acid), PFD 2-3-2-2 (ursolic acid) isolated from the perilla leaves, tormentic acid purchased as a standard, and Rosmarinic acid purchased separately.

The degree of Th17 differentiation inhibition was evaluated by measuring the expression level of IL-17A after treating cells differentiated with IL-6 and TGF-β with PFD 4-2-5-6, PFD 2-3-2-2, standard tomentic acid, or rosmarinic acid.

In Figures 9 and 10, Th0 refers to the group that was not treated with IL-6 and TGF-β for differentiation. In addition, in Figures 9 and 10, Th17 refers to the group that was maximally differentiated by adding only IL-6 and TGF-β.

Figure 9 shows the IL-17A expression rate (%) measured using PFD 4-2-5-6 (tormentic acid) and rosmarinic acid isolated from perilla leaves. Rosmarinic acid, which has the highest content in the DCM layer of perilla leaves, was also measured for its effect as an indicator component for extract standardization.

More specifically, Fig. 9 shows the IL-17A expression rate (%) measured at concentrations of 1, 5, and 10 ㎍/mL of PFD 4-2-5-6 (tormentic acid) isolated from the perilla leaves and rosmarinic acid purchased separately from Sigma.

From Figure 9, it was confirmed that tomentic acid and rosmarinic acid isolated from the perilla leaves inhibited IL-17A expression, and thus the Th17 suppression efficacy could be confirmed.

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

More specifically, Fig. 10 shows the IL-17A expression rate (%) measured using 5, 10, 20, and 50 μg/mL of perilla leaf ethanol extract, 1, 2, and 5 μg/mL of PFD 4-2-5-6 (tormentic acid) isolated from perilla leaves, 1, 2, and 5 μg/mL of tormentic acid standard, and 0.5, 1, and 2 μg/mL of PFD 2-3-2-2 (ursolic acid) isolated from perilla leaves.

From Fig. 10, it was confirmed that tomentic acid (PFD 4-2-5-6) isolated from the ethanol extract of perilla leaves inhibited IL-17A expression, and thus its Th17 suppression efficacy could be confirmed.

PFD 4-2-5-6 (tormentic acid) isolated from perilla leaves showed a significant inhibitory effect on IL-17A expression at 10 ㎍/mL, while tomentic acid purchased as a standard showed a significant inhibitory effect at 5 and 10 ㎍/mL. Since the scales for the inhibition ratios did not differ significantly, it was confirmed that PFD 4-2-5-6 isolated from perilla leaves and tomentic acid purchased as a standard showed the same degree of efficacy.

Test Example 5. Measurement of the effect of tomentose isolated from perilla leaves in a model of inflammatory enteritis induced by dextran sulfate sodium (DSS)

7-week-old C57BL/6 (male) mice were purchased from Orient Bio and acclimatized under SPF conditions (IACUC number: #202000120). To induce intestinal inflammation, drinking water containing 2.5% DSS was provided for 7 days, and water consumption was checked daily (the experiment was conducted by dividing the mice into 6 groups of 6 each).

The vehicle (corn oil) treatment group served as a negative control group. In addition, the group treated only with DSS (DSS-induced and drug-untreated group) served as a positive control group.

The test groups were as follows: DSS and 50 mg/kg/day ethanol extract of perilla leaves treatment group, DSS and 25 mg/kg/day tomentoic acid treatment group, DSS and 50 mg/kg/day tomentoic acid treatment group, and DSS and mesalazine (50 mg/kg/day p.o.) treatment group.

After 7 days, the water containing 2.5% DSS was replaced with water, and 1 day later (day 8), the animals were sacrificed, and the spleen, mesenteric lymph nodes (MLN), and intestine were removed for experiments.

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

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

Figure 12A shows the results of measuring the change in intestinal length after treatment with a vehicle treatment group (Control), a DSS-induced and drug-free group (DSS), a DSS and 50 mg/kg/day perilla leaf ethanol extract treatment group (P.F extracts 50 mg/kg), a DSS and 25 mg/kg/day tomentic acid treatment group (Tormentic acid 25 mg/kg), a DSS and 50 mg/kg/day tomentic acid treatment group (Tormentic acid 50 mg/kg), and a DSS and 50 mg/kg/day mesalazine treatment group (Mesalazine 50 mg/kg). In addition, Figure 12B shows this as a graph.

The more severe the intestinal inflammation, the shorter the intestinal length, and the closer it is to the control group, the more the intestinal inflammation is improved.

As shown in Figures 12A and 12B, the group treated with DSS only (DSS) showed a significant decrease in intestinal length compared to the vehicle-treated group (Control), whereas in the case of mice administered 25 mg/kg of tomentose, the decrease in intestinal length caused by DSS was significantly suppressed.

In addition, as shown in Figures 12A and 12B, tomentic acid isolated from the perilla leaf extract significantly inhibited the decrease in intestinal length caused by inflammatory enteritis induced by DSS.

Figures 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 histological photographs of the colon in mice measured by H&E staining. It can be observed that the mucosa layer of the DSS-treated group was destroyed compared to the vehicle-treated group (Control), whereas in the group co-treated with 25 mg/kg of tomentose, slight edema was observed, but the destruction of the mucosa layer was significantly improved compared to the DSS-treated group.

Additionally, Figure 13B numerically displays the severity of pathological symptoms in the stained tissue.

Figures 13A and 13B show that tomentose isolated from the perilla leaves significantly inhibited the histological changes of the intestine induced by DSS.

In addition, the histological changes in the intestine induced by DSS were more suppressed in the groups treated with 25 mg/kg or 50 mg/kg of tomentose than in the group treated with 50 mg/kg of mesalazine.

Test Example 6. Evaluation of Cytokine Expression Inhibition

Figures 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.

That is, CD4 ⁺ T cells were isolated from the spleen, MLN, and LPL of each experimental mouse, stimulated with PMA, ionomycin, and Golgi-plug for 4 hours, and then CD4 ⁺ IL-17A ⁺ was analyzed using flow cytometry.

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

Statistical analysis

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

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

Those skilled in the art will appreciate that the present invention can be implemented in modified forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated by the claims, not the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (16)

A pharmaceutical composition comprising a compound represented by the following chemical formula 1 isolated from a perilla leaf extract or a salt thereof as an active ingredient, and used for preventing or treating an autoimmune disease selected from rheumatoid arthritis and inflammatory bowel disease by inhibiting IL-17A expression:
[Chemical Formula 1]
. A pharmaceutical composition according to claim 1, wherein the extraction solvent of the perilla leaf extract is water, a C ₁ to C ₄ alcohol, or a mixture thereof. A pharmaceutical composition according to claim 1, wherein the compound is separated by adding at least one solvent selected from the group consisting of water, C ₁ to C ₅ straight-chain or branched alcohol, hexane, dichloromethane, and ethyl acetate to a perilla leaf extract. A pharmaceutical composition according to claim 1, wherein the compound is separated by adding at least one solvent selected from the group consisting of hexane, dichloromethane, ethyl acetate, butanol, and water to a perilla leaf extract. A pharmaceutical composition according to claim 1, wherein the compound is separated by sequentially adding hexane, dichloromethane, ethyl acetate, butanol and water to an ethanol extract of perilla leaves. A pharmaceutical composition according to claim 5, wherein the separation is performed by fractionating with dichloromethane and separating the obtained dichloromethane layer through silica gel column chromatography. A pharmaceutical composition according to claim 6, wherein the silica gel column chromatography method separates the composition through a gradient method of a solvent. A pharmaceutical composition 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. A pharmaceutical composition according to claim 8, 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 at least one selected from the group consisting of citric acid, acetic acid, lactic acid, tartariac 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, glutamic acid, and aspartic acid. A pharmaceutical composition according to claim 1, wherein the pharmaceutical composition comprises a diluent or a carrier. A pharmaceutical composition according to claim 1, formulated as an oral preparation or a parenteral preparation. A pharmaceutical composition according to claim 11, wherein the oral preparation is a tablet, a capsule, a pill, a powder, a granule, a suspension or a syrup, and the parenteral preparation is a cream, a lotion, an ointment, a solution, a gel, a cataplasma, a patch, an aerosol, a fluid extract, an elixir, a needle, a sachet or an injection. A step of extracting the perilla leaves with 50% (v/v) to 100% (v/v) ethanol;
A step of fractionating the ethanol extract obtained from the above step into 30% (v/v) to 99% (v/v) dichloromethane; and
A step of obtaining a compound of chemical formula 1 of claim 1 by separating the dichloromethane fraction obtained from the above step using silica gel column chromatography; comprising:
A method for producing a pharmaceutical composition used for preventing or treating an autoimmune disease selected from rheumatoid arthritis and inflammatory bowel disease by inhibiting IL-17A expression according to claim 1. A food composition comprising a compound represented by the following chemical formula 1 or a salt thereof isolated from a perilla leaf extract as an effective ingredient and used for preventing or improving an autoimmune disease selected from rheumatoid arthritis and inflammatory bowel disease by inhibiting IL-17A expression:
[Chemical Formula 1]
.
A pharmaceutical composition according to claim 1, wherein the autoimmune disease is inflammatory bowel disease. A pharmaceutical composition according to claim 1, wherein the inflammatory bowel disease is Crohn's disease.