KR20230052446A - Composition for Preventing or Treating Inflammatory Diseases Comprising Metabolites from Antarctic Fungal Strain Pleosporales sp. SF-7343 - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating inflammatory diseases and a food for improving inflammation, containing a metabolic compound derived from Antarctic fungi, and more specifically, to a pharmaceutical composition for preventing or treating inflammatory diseases and a food for improving inflammation, containing a metabolic compound derived from Antarctic fungal strain Pleosporales sp. SF-7343. According to the present invention, the composition containing a metabolic compound derived from Antarctic fungal strain Pleosporales sp. SF-7343 inhibits the production of pro-inflammatory cytokines, chemokines and mediators in HaCaT cells stimulated with TNF-α / IFN-γ. Accordingly, the composition can be used for the prevention or treatment of inflammatory diseases and can especially control skin inflammation such as atopic dermatitis.

Description

The Antarctic fungal strain Pleosporeles sp. Composition for Preventing or Treating Inflammatory Diseases Comprising Metabolites from Antarctic Fungal Strain Pleosphorales sp. SF-7343}

The present invention relates to a pharmaceutical composition for preventing or treating inflammatory diseases containing a metabolite derived from an Antarctic fungus and a food for improving inflammation, and more particularly, to a pharmaceutical composition comprising an Antarctic fungus Pleossporales sp. It relates to a pharmaceutical composition for preventing or treating inflammatory diseases and a food for improving inflammation, including a metabolite derived from SF-7343.

Atopic dermatitis (AD) is a chronic inflammatory skin disease, the cause of which is complex and has multiple factors. The prevalence of atopic dermatitis is quite high at 20% in children and 10% in adults. Common symptoms of AD include dry, itchy skin, eczema and redness. In addition, AD is associated with other atopic disorders, skin ulcers, and sleep disorders, thereby reducing quality of life. From an immunological point of view, AD is a helper (Th) 2 cell mediated skin disease. It occurs due to an imbalance between the levels of Th1 and Th2 cells (Woods, C, A., Am J Manag Care 2017 , 23 , 115-123). Some inflammatory cytokines and chemokines secreted by Th2 cells can affect skin cells such as keratinocytes and mast cells. When mast cells are activated, histamine and many other cytokines and chemokines are released, promoting the infiltration of immune cells into inflammatory lesions. At the lesion site, additional inflammation-related skin barrier function degradation, hypersensitivity enhancement, and scratch-related irritation exacerbate eczema, causing a vicious cycle of inflammation. It also induces overproduction of immunoglobulin E (IgE), which in turn accelerates the progression of AD.

Keratinocytes are the most abundant cells in the epidermis and represent the first line of the host defense system. These cells form a physical skin barrier by sensing pathogens through innate immune receptors, initiating antibacterial responses, and producing various cytokines, chemokines, and antimicrobial peptides (Boguniewicz, M., Leung, D, Y, G. Immunol Rev 2011 , 242 , 233-246). In the epidermal barrier damaged by AD, penetration of latent allergens and pathogens activates keratinocytes. Among the dysregulation of the immune response in AD, activated keratinocytes play an important role in several biological processes contributing to AD pathogenesis (Wilson, V, G., Methods Mol Biol 2014 , 1195 , 33-41).

Meanwhile, because of Antarctica's unique location and climate, its microbes are well adapted and able to withstand extreme conditions. Fungi play an important role in finding structurally unique secondary metabolites. A number of studies have shown that Antarctic fungi produce bioactive metabolites (Barnes, D, K, A, Clarke, A., Curr Biol 2011 , 21 , 451-457). Secondary metabolites derived from Antarctic fungi can be classified into alkaloids, terpenoids, polyketides, peptides, and sterols. Therefore, Antarctic fungi are an attractive source for the isolation of novel secondary metabolites with novel structures. However, previous studies have only reported that alternariol inhibits LPS-induced inflammation in THP-1-derived macrophages (Kollarova, J, et al., Arch Toxicol 2018 , 92 , 3347-3358). However, the fact that alternariol and altenuene have anti-inflammatory effects has not been reported yet.

Accordingly, the present inventors have made diligent efforts to screen metabolites with excellent anti-inflammatory activity, and as a result, the Antarctic fungus Pleossporales sp. Metabolic compounds were isolated from SF-7343, and it was confirmed that the metabolites had anti-inflammatory effects on the inflammatory response induced by TNF-α/IFN-γ in HaCaT cells, thereby completing the present invention.

The above information described in this background section is only for improving the understanding of the background of the present invention, and therefore does not include information that forms prior art known to those skilled in the art to which the present invention belongs. may not be

An object of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory diseases and a food for improving inflammation, including a metabolite derived from Antarctic fungi having excellent anti-inflammatory activity.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases comprising one or more compounds selected from the group consisting of Formulas 1 to 3 below.

[Formula 1]

[Formula 2]

[Formula 3]

Here, the R ₁ to R ₆ are each independently H, straight-chain or branched-chain C _1-10 alkyl, C _1-10 alkoxy, C _3-10 cycloalkyl, C _2-10 alkenyl or C _6-10 it is aryl

The present invention also provides a food for improving inflammation containing at least one compound selected from the group consisting of Formulas 1 to 3 above.

According to the present invention, the Antarctic fungus Pleosporeles sp. Since the composition containing metabolites derived from SF-7343 inhibits the production of pro-inflammatory cytokines, chemokines and mediators in HaCaT cells stimulated with TNF-α/IFN-γ, preventing or treating inflammatory diseases It can be usefully used for various purposes, and in particular, it can control skin inflammation such as atopic dermatitis.

Figure 1 depicts the cytotoxicity of compounds 1-3. Cytotoxicity was evaluated in cells treated at the indicated concentrations for 24 hours. Data are presented as mean ± SD values of three independent experiments. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. control group.
Figure 2 shows the effect of compounds 1-3 on IL-6 secretion in TNF-α/IFN-γ stimulated HaCaT cells. IL-6 secretion was measured using the culture supernatant of HaCaT cells stimulated with TNF-α/IFN-γ. Cells were pretreated with the indicated concentrations of compounds 1-3 for 3 hours and then stimulated with TNF-α/IFN-γ for 24 hours. Data are presented as the mean ± SD of three independent experiments. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. Control. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. TNF-α/IFN-γ-treated group.
Figure 3 shows the effect of compounds 1-3 on IL-8, MDC and RANTES secretion in TNF-α/IFN-γ stimulated HaCaT cells. Levels of IL-8, MDC and RANTES were measured using culture supernatants of HaCaT cells stimulated with TNF-α/IFN-γ. Cells were pretreated with the indicated concentrations of Compound 1 and Compound 2 for 3 hours and then stimulated with TNF-α/IFN-γ for 24 hours. Data are presented as the mean ± SD of three independent experiments. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. Control. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. TNF-α/IFN-γ-treated group.
Figure 4 shows the effect of Compound 1 and Compound 2 on TNF-α/IFN-γ-induced expression of ICAM-1 in HaCaT cells. Expression of ICAM-1 was measured using Western blotting. Cells were pretreated with the indicated concentrations of compounds 1 and 2 for 3 hours and then stimulated with TNF-α/IFN-γ for 24 hours. Data are presented as the mean ± SD of three independent experiments. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. Control. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. TNF-α/IFN-γ-treated group.
Figure 5 shows the effect of Compound 1 and Compound 2 on the expression of FLG and IVL in HaCaT cells. Expression of FLG and IVL was measured using cell lysis. Cells were pretreated with the indicated concentrations of compounds 1 and 2 for 3 hours and then stimulated with TNF-α/IFN-γ for 24 hours. Data are presented as the mean ± SD of three independent experiments. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. Control. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. TNF-α/IFN-γ-treated group.
6A and 6B show the effects of Compound 1 and Compound 2 on NF-κB, p-STAT1 and p-STAT3 signaling pathways in HaCaT cells. Cells were pre-treated with the indicated concentrations of compounds 1 and 2 for 3 hours and then stimulated with TNF-α/IFN-γ for 15 minutes. Cytosol and nuclear extracts were separated, and the levels of p65, p-IκBα and IκBα were partially determined by Western blotting. Expression of p-stat3, stat3, p-stat1, and stat1 and total protein were measured using Western blotting. The bar graph represents the quantitative density of bands. Data are presented as the mean ± SD of three independent experiments. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. Control. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. TNF-α/IFN-γ-treated group.
7A and 7B show the effect of compounds 1 and 2 on heme oxygenase (HO)-1 expression and Nrf2 translocation. HaCaT cells were cultured for 12 hours with the indicated concentrations of compound 1 (20-60 μM) and compound 2 (2.5-10 μM) (AD). HaCaT cells were incubated with 60 μM of Compound 1 for 0-24 hours (E,F). HaCaT cells were treated with 60 μM compound 1 for 0.5, 1, 1.5 hours (G,H). Nuclei were fractionated and Western blot analysis for HO-1 and Nrf2 expression was performed. Data are presented as the mean ± SD of three independent experiments. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. Control. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. TNF-α/IFN-γ-treated group.
8 shows the effect of Compound 1 on HaCaT cells stimulated with TNF-α/IFN-γ. HaCaT cells were pretreated with compound 1 (60 μM) in the presence or absence of SnPP (40 μM) for 3 h and stimulated with TNF-α/IFN-γ for 24 h to measure IL-6, IL-8, and RANTES or 15 NF-κB binding activity was measured over a period of minutes. Levels of IL-6, IL-8 and RANTES and NF-κB binding activity were detected by ELISA. Data are presented as the mean ± SD of three independent experiments. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. Control. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. TNF-α/IFN-γ-treated group. ^Δp < 0.05, ^ΔΔ p < 0.01 , ^ΔΔΔ p < 0.001 vs. Compound 1 +TNF-α/IFN-γ-treated group.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is one well known and commonly used in the art.

In one embodiment of the present invention, the Antarctic fungus Pleosporeles sp. Metabolites of compounds 1 to 3 were isolated from the extract of SF-7343 (accession number: KCTC 14724BP), and their structures were confirmed. In addition, the metabolites (Compounds 1 to 3) inhibit the secretion of IL-6, IL-8, MDC and RANTES in HaCaT cells stimulated with TNF-α/IFN-γ, and inhibit the secretion of ICAM-1, FLG and IVL. By suppressing the expression and suppressing the activation of NF-κB, p-STAT1 and p-STAT3, it was confirmed to have an effect of preventing or treating inflammatory diseases.

Accordingly, in one aspect, the present invention relates to a pharmaceutical composition for preventing or treating inflammatory diseases comprising at least one compound selected from the group consisting of Formulas 1 to 3 below.

[Formula 1]

[Formula 2]

[Formula 3]

Here, the R ₁ to R ₆ are each independently H, straight-chain or branched-chain C _1-10 alkyl, C _1-10 alkoxy, C _3-10 cycloalkyl, C _2-10 alkenyl or C _6-10 it is aryl

In the present invention, the C _1-10 alkyl group means a straight or branched chain saturated hydrocarbon group having 1 to 10 carbon atoms, and examples thereof include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert -Butyl, isobutyl, n-pentyl, n-hexyl, 1-ethylpropyl, 2,2-dimethylpropyl, etc. are mentioned.

In the present invention, the C _1-10 alkoxy group means a straight-chain or branched-chain alkyloxy group having 1 to 10 carbon atoms, and examples thereof include methoxy, ethoxy, propoxy, prop-2-oxy, butoxy , but-2-oxy, methyl prop-2-oxy, isopropoxy, tert-butoxy and the like.

In the present invention, a C _3-10 cycloalkyl group means a saturated hydrocarbon ring having 3 to 10 carbon atoms, and is exemplified by cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc. In addition to monocycloalkyl groups, Polycycloalkyl groups, such as bicycloalkyl groups and tricycloalkyl groups, are also included, and bicycloalkyl groups include norbornyl, such as exo-2-norbornyl, endo-2-norbornyl, 3 -Pynanyl, bicyclo[3.1.0]hexyl group, bicyclo[2.2.1]heptyl group, bicyclo[2.2.2]octo-2-yl group, etc., tricycloalkyl group is adamantyl, for example , 1-adamantyl group, 2-adamantyl group, etc. are mentioned.

In the present invention, a C _2-10 alkenyl group is a straight-chain or branched-chain hydrocarbon radical containing at least one double bond and having 2 to 10 carbon atoms, such as ethenyl, 2-propenyl, 3-part tenyl, 2-butenyl, 2-pentenyl, 3-pentenyl, 3-methyl-2-butenyl or 3-hexenyl; and the like.

In the present invention, the C _6-10 aryl group means an aryl group having 6 to 10 carbon atoms, and examples thereof include a phenyl group, a naphthyl group, an anthryl group, and a phenanthryl group.

In the present invention, the R ₁ to R ₆ may each independently represent H, CH ₃ or CH ₂ CH ₃ , preferably R ₁ is H or CH ₃ , and R ₂ to R ₆ are Each is CH ₃ .

In the present invention, R ₁ is CH ₃ and R ₂ and R ₃ are each CH ₃ The metabolite of Formula 1 is defined as alternate C, wherein R ₁ is H, and R ₂ and R ₃ are each CH ₃ . The metabolite of Formula 1 is defined as altenusin, and the term “Compound 1” refers to a metabolite in Formula 1 wherein R ₁ is CH ₃ or H, and R ₂ and R ₃ are CH ₃ respectively.

In the present invention, the metabolite of Chemical Formula 2 in which R ₄ is CH ₃ is defined as alternariol, and the term “Compound 2” refers to a metabolite in which R ₄ is CH ₃ in Chemical Formula 2.

In the present invention, the metabolite of Formula 3 in which R ₅ and R ₆ are each CH ₃ is defined as altenuene, and the term "Compound 3" in Formula 3 wherein R ₅ and R ₆ are each CH ₃ means a metabolite.

In the present invention, the metabolite is an Antarctic fungus Pleosporeles sp. It can be characterized as isolated from SF-7343.

In the present invention, the term "anti-inflammatory" means an action that inhibits or reduces inflammation.

In the present invention, the term "inflammation" is one of the defense reactions of biological tissues to certain stimuli, and is a biological defense mechanism to restore the original state by removing injuries caused by various harmful stimuli. Stimulation of inflammation includes infection or chemical and physical stimulation, and the process of inflammation can be divided into two types: acute and chronic inflammation. Acute inflammation is a short-term reaction within a few days, and plasma components or blood cells enter the microcirculatory system and are involved in the removal of foreign substances. Chronic inflammation lasts for a long time, and tissue proliferation is seen.

In the present invention, the term "inflammatory disease" is a general term for diseases in which inflammation is the main lesion.

In the present invention, the inflammatory disease is arthritis, rhinitis, hepatitis, keratitis, gastritis, enteritis, nephritis, bronchitis, pleurisy, peritonitis, spondylitis, pancreatitis, inflammatory pain, urethritis, cystitis, burn inflammation, dermatitis, periodontitis, gingivitis and degenerative It may be characterized in that it is selected from the group consisting of neuroinflammation, but is not limited thereto, preferably dermatitis, more preferably atopic dermatitis.

In the present invention, the pharmaceutical composition may be characterized by having one or more of the following properties.

1) inhibition of secretion of IL-6, IL-8, MDC (macrophage-derived chemokine) and RANTES;

2) inhibition of expression of ICAM-1;

3) inhibition of expression of filaggrin (FLG) and involcurin (IVL);

4) inhibition of activation of NF-κB, p-STAT1 and p-STAT3; and

5) Promotion of HO-1 expression and Nrf2 translocation.

In one embodiment of the present invention, it was confirmed that Compound 1 and Compound 2, in which R ₁ is CH ₃ , exhibit anti-inflammatory effects by reducing the secretion of IL-6, IL-8, MDC, and RANTES (Examples 3 and 4 ). Keratinocytes react with pro-inflammatory cytokines such as TNF-α and IFN-γ, which are involved in the expression of numerous inflammatory mediators in chronic inflammatory skin diseases such as psoriasis and AD. Activated keratinocytes release pro-inflammatory cytokines and chemokines that play an important role in the infiltration of inflammatory immune cells into AD lesions. As a result, levels of some pro-inflammatory cytokines and chemokines are increased in inflammatory AD lesions. IL-6, as a pro-inflammatory cytokine, is involved in epidermal proliferation of psoriatic epithelium, affects the function of dermal inflammatory cells, and induces differentiation of human Th17 cells. Chemokines such as IL-8, RANTES, TARC and MDC play important roles in mediating leukocyte recruitment from the blood to the skin and establishing the microenvironment. Therefore, in the present invention, pro-inflammatory cytokines and chemokines were targeted to treat various skin diseases resistant to existing therapies.

In another example of the present invention, it was confirmed that compounds 1 and 2 in which R ₁ is CH ₃ up-regulated the expression of IVL, and compound 1 up-regulated the expression of FLG (Example 6). Increased pro-inflammatory cytokines and chemokines induce changes in skin barrier proteins, which are one of the major early factors in the pathogenesis of AD. Some proteins, such as FLG and IVL, which play important roles in keratinocyte differentiation and skin barrier function, resist at the cell surface. Cytokines secreted by keratinocytes and other skin-inhabited cells alter the barrier function of the skin by intercellular communication. For example, cytokines manipulate keratinocyte proliferation and differentiation by regulating gene expression within these cells. Unregulated cytokine expression can lead to dysfunction of the epidermal barrier, as seen in diseases such as AD and psoriasis. Co-stimulation with TNF-α/IFN-γ for 24 h reduced FLG and IVL, suggesting that the skin barrier function was partially impaired.

In another embodiment of the present invention, it was confirmed that Compound 1 and Compound 2, in which R ₁ is CH ₃ , have an inhibitory effect on the expression of inflammatory cytokines and chemokines through inhibition of STAT1, STAT3 and NF-κB signaling pathways (Example Example 7). Activation of STAT and NF-κB is important for increasing AD-related inflammatory gene expression in HaCaT cells. When STAT1, STAT3, and NF-κB are activated by pro-inflammatory cytokines, STAT1, STAT3, and NF-κB translocate from the cytoplasm to the nucleus of keratinocytes, resulting in the expression of inflammatory mediators. It was confirmed that administration of Compound 1 and Compound 2 to HaCaT cells stimulated with TNF-α and IFN-γ attenuated the increased secretion of inflammatory mediators and inhibited STAT1 and STAT3 phosphorylation, IκBα degradation, and NF-κB nuclear translocation.

In another example of the present invention, it was confirmed that Compound 1, wherein R ₁ is CH ₃ , significantly induced the expression of HO-1 and promoted the translocation of Nrf2 from the cytoplasm to the nucleus (Example 8). Many studies have shown that various bioactive compounds exert anti-inflammatory activity by inducing the expression of HO-1 in inflammatory disease models (Lee, J, H., et al., Front Pharmacol 2020 , 11 , 1018.). HO-1 is the rate-limiting enzyme for heme breakdown and produces ferrous iron, carbon monoxide and biliverdin. These three by-products exhibit a variety of beneficial effects including anti-inflammatory, antioxidant and cytoprotective effects in many pathological conditions. Several studies have shown that HO-1 expression exerts immunomodulatory effects on inflammatory skin diseases such as AD (Wu, W, Q, et al., Mol Med Rep 2019, 20 , 1761-1771). When Nrf2, a transcription factor, is activated by binding to Keap1 in the cytoplasm, Nrf2 is detached from Keap1 and translocated to the nucleus, where it binds to antioxidant response elements (ARE) to upregulate the expression of antioxidant enzymes including HO-1. induce protection.

In the present invention, the pharmaceutical composition may be characterized in that it further comprises a pharmaceutically acceptable carrier, excipient or diluent.

Carriers, excipients and diluents that may be included in the composition include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil. When formulating the composition, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

The pharmaceutical composition according to the present invention may be formulated and used in various forms according to conventional methods. Suitable formulations include tablets, pills, powders, granules, dragees, hard or soft capsules, solutions, suspensions or emulsions, injections, oral formulations such as aerosols, external preparations, suppositories, and sterile injection solutions. It is not limited to this.

The pharmaceutical composition according to the present invention can be prepared in a suitable dosage form using a pharmaceutically inert organic or inorganic carrier. That is, when the dosage form is a tablet, coated tablet, dragee and hard capsule, it may contain lactose, sucrose, starch or a derivative thereof, talc, calcium carbonate, gelatin, stearic acid or a pharmaceutically acceptable salt thereof. In addition, if the dosage form is a soft capsule, it may contain vegetable oil, wax, fat, semi-solid and liquid polyol. In addition, when the formulation is in the form of a solution or syrup, water, polyol, glycerol, and vegetable oil may be included.

The "pharmaceutically acceptable salt" means a formulation of a compound that does not cause serious irritation to the organism to which the compound is administered and does not impair the biological activity and physical properties of the compound. The pharmaceutical salt is an acid that forms a non-toxic acid addition salt containing a pharmaceutically acceptable anion, for example, inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, tartaric acid, formic acid, citric acid , organic carbonic acids such as acetic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, salicylic acid, etc., sulfonic acid such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid Acid addition salts formed with phonic acids and the like are included. For example, pharmaceutically acceptable carboxylic acid salts include metal salts or alkaline earth metal salts formed by lithium, sodium, potassium, calcium, magnesium, etc., amino acid salts such as lysine, arginine, guanidine, dicyclohexylamine, N organic salts such as -methyl-D-glucamine, tris(hydroxymethyl)methylamine, diethanolamine, choline and triethylamine; and the like. Acids such as oxalic acid are not pharmaceutically acceptable, but as intermediates for obtaining pharmaceutically acceptable salts, they can be used in the preparation of useful salts.

The pharmaceutical composition according to the present invention may further include a preservative, a stabilizer, a wetting agent, an emulsifier, a solubilizing agent, a sweetening agent, a coloring agent, an osmotic pressure regulator, an antioxidant, and the like, in addition to the above carriers.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is the type, severity, and activity of the drug of the patient's disease , sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

The composition of the present invention can be used in conjunction with the treatment of inflammatory diseases such as immunotherapy, chemotherapy and radiation therapy, which can be recognized by those skilled in the art as being effective in preventing or treating inflammatory diseases, and pharmaceutical compositions for treating other inflammatory diseases. .

The pharmaceutical composition of the present invention can be administered to a subject by various routes. The mode of administration may be, for example, by subcutaneous, intravenous, intramuscular or intrauterine intrathecal or intracerebrovascular injection. The pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient, together with various related factors such as the disease to be treated, the route of administration, the patient's age, sex and weight, and the severity of the disease.

In another aspect, the present invention relates to a food for improving inflammation comprising at least one compound selected from the group consisting of Formulas 1 to 3 above.

In the present invention, R ₁ to R ₆ of the metabolite may be each independently H, CH ₃ or CH ₂ CH ₃ , preferably R ₁ is H or CH ₃ , and R ₂ to R ₆ is each CH ₃ .

In the present invention, the term "food" refers to meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, There are vitamin complexes, health functional food and health food, etc., and includes all foods in a conventional sense.

The health functional (sex) food (functional food) is the same term as food for special health use (FoSHU), and is a medicine processed to efficiently display bioregulatory functions in addition to nutrient supply, and has high medical effect. means food. Here, "function (sex)" means to obtain useful effects for health purposes such as regulating nutrients for the structure and function of the human body or physiological functions. The food of the present invention can be prepared by a method commonly used in the art, and can be prepared by adding raw materials and ingredients commonly added in the art during the preparation. In addition, the formulation of the food may be prepared without limitation as long as the formulation is recognized as food, and the health functional food according to the present invention may be in the form of powder, granule, tablet, capsule or beverage.

The health food (health food) means a food that has an active health maintenance or promotion effect compared to general food, and health supplement food (health supplement food) means food for the purpose of health supplement. In some cases, the terms health functional food, health food, and health supplement food are used interchangeably.

The food composition may further include a physiologically acceptable carrier. The type of carrier is not particularly limited, and any carrier commonly used in the art may be used.

In addition, the composition may include additional ingredients that are commonly used in food and can improve smell, taste, and sight. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid, and the like may be included. In addition, minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), and chrome (Cr) may be included. In addition, amino acids such as lysine, tryptophan, cysteine, and valine may be included.

In addition, the composition contains preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), disinfectants (bleaching powder and high bleaching powder, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisole (BHA), butylhydroxy toluene (BHT), etc.), coloring agents (tar colorant, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), bleaching agents (sodium sulfite), seasonings (MSG, etc.), sweeteners (dulcin, cyclemate, saccharin, sodium, etc.) ), flavoring (vanillin, lactones, etc.), leavening agent (alum, D-potassium hydrogentartrate, etc.), strengthening agent, emulsifier, thickener (thickener), coating agent, gum base agent, foam inhibitor, solvent, food additives such as improver ) may be included. The additive may be selected according to the type of food and used in an appropriate amount.

Pleosporeles sp. of the present invention. Along with the extract, it may further include food additives acceptable for food science, may be used together with other foods or food ingredients, and may be appropriately used according to conventional methods. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment).

In the present invention, the terms "improvement", "prevention" and "treatment" should be interpreted in the broadest sense, and "improvement" refers to any action that temporarily/continuously alleviates a disease or one or more clinical symptoms. "Prevention" means preventing one or more of the clinical symptoms of a disease from progressing in a patient who may be exposed to or predisposed to a disease, but who has not yet experienced or manifested symptoms of the disease. "Treatment" means any action that prevents or reduces the development of a disease or one or more clinical symptoms thereof.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for exemplifying the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example

Example 1: Structure Determination of Isolated Metabolites

The cultured fungal strain Pleosporeles sp. The dried extract of SF-7343 went through several chromatographic steps to obtain alternate C (Compound 1 where R ₁ is CH ₃ ), altenusin (Compound 1 where R ₁ is H), alternariol (Compound 2), and altenuene (Compound 3). did Their structures were determined based on 1D and 2D NMR, MS analysis and compared with data reported in the literature.

Example 2: Effect of compounds 1 to 3 on cell viability

The cytotoxicity of compounds 1 to 3 on HaCaT cells was evaluated using the MTT assay. The results are shown in Figure 1. For subsequent experiments, cells were treated with these four compounds in a safe concentration range: Compound 1 in which R ₁ is CH ₃ (20-60 μM), Compound 1 in which R ₁ is H ( 5-20 μM), compound 2 (2.5-10 μM) and compound 3 (20-80 μM).

Example 3: Effects of Compounds 1 to 3 on IL-6 secretion in HaCaT cells stimulated with TNF-α/IFN-γ

In order to confirm the anti-inflammatory effect of compounds 1-3 in HaCaT cells stimulated with TNF-α/IFN-γ, IL-6 secretion was analyzed first. As shown in FIG. 2, compounds 1 and 2 in which R ₁ is CH ₃ significantly inhibited IL-6 secretion, and compound 1 in which R ₁ is H showed an inhibitory effect on IL-6 secretion at high concentrations. . Compound 3 did not significantly affect IL-6 secretion. Among the four compounds, compound 1 and compound 2, in which R ₁ is CH ₃ , showed the most remarkable effect of inhibiting IL-6 secretion in HaCaT cells. Therefore, in the following further experiments, the two most effective compounds were tested.

Example 4: Effect of Compound 1 and Compound 2 on Secretion of IL-8, MDC and RANTES in HaCaT Cells Stimulated with TNF-α/IFN-γ

The epidermis is not only a physical barrier, but also a chemical and immunological barrier that produces various cytokines and chemokines. The effects of Compound 1 and Compound 2, wherein R ₁ is CH ₃ , on the following inflammatory cytokines and chemokines are shown in FIG. 3 . IL-8, macrophage-derived chemokine (MDC) and RANTES (regulated upon activation, normal T cell expressed and presumably secreted) levels were determined from cell culture supernatants using ELISA. The secretion of IL-8, MDC, and RANTES was significantly increased in the TNF-α/IFN-γ stimulation group compared to the control group, and decreased in a dose-dependent manner in the groups pretreated with Compound 1 and Compound 2 where R ₁ is CH ₃ .

Example 5: Effect of Compound 1 and Compound 2 on TNF-α/IFN-γ Induced ICAM-1 Expression in HaCaT Cells

In inflammatory conditions, keratinocytes can also express adhesion molecules such as ICAM-1. Expression of ICAM-1 promotes the adsorption of leukocytes to the surrounding skin tissue. The effects of Compound 1 and Compound 2 on TNF-α/IFN-γ-induced ICAM-1 expression were tested. As shown in Fig. 4, both compound 1 and compound 2, wherein R ₁ is CH ₃ , significantly reduced ICAM-1 expression in a dose-dependent manner.

Example 6: Effect of Compound 1 and Compound 2 on FLG and IVL Expression in HaCaT Cells

Filaggrin (FLG) and involcurin (IVL) are two proteins that encode genes present in the uppermost layer of the epidermis, and play an important role in keratinocyte differentiation and skin barrier function. In inflammatory keratinocytes, the expression of these two proteins is reduced and the skin barrier function is weakened. It was confirmed that when compound 1 in which R ₁ is CH ₃ was treated, the decrease in FLG and IVL expression was recovered compared to the TNF-α/IFN-γ group (FIGS. 5A-C). Compared to the TNF-α/IFN-γ group, Compound 2 suppressed the decrease in IVL expression induced by TNF-α/IFN-γ in HaCaT cells (FIGS. 5D and F).

κB, p-STAT1 및 p-STAT3 신호 전달 경로에 대한 화합물 1 및 화합물 2의 효과 Example 7: NF- in HaCaT cells

Effect of compound 1 and compound 2 on κB, p-STAT1 and p-STAT3 signaling pathways

B, p-STAT1 및 p-STAT3 신호 전달 경로를 조절할 수 있음을 알 수 있다.Simultaneous stimulation of TNF-α and IFN-γ in keratinocytes leads to the release of cytokines and chemokines, a process regulated by the NF-κB, p-STAT1 and p-STAT3 signaling pathways. As shown in Fig. 6A-D, when cells were treated with TNF-α/IFN-γ, p-IκBα and p65 were significantly increased compared to the control group. IκBα phosphorylation and p65 activation were decreased when pretreatment with Compound 1 and Compound 2, in which R ₁ is CH ₃ , was performed. The ratio of p-IκBα/IκBα was also decreased. Also, as shown in FIGS. 6E-H, compounds 1 and 2 in which R ₁ is CH ₃ markedly downregulated STAT1 and STAT3 phosphorylation. From these results, compounds 1 and 2, in which R ₁ is CH ₃ , inhibited NF-κ in TNF-α/IFN-γ-treated HaCaT cells.

It can be seen that the B, p-STAT1 and p-STAT3 signaling pathways can be regulated.

Example 8: Effects of Compound 1 and Compound 2 on HO-1 expression and Nrf2 translocation

To investigate whether the compounds induce expression of heme oxygenase (HO)-1, HaCaT cells were treated with Compound 1 (20-60 μM) and Compound 2 (2.5-10 μM), where R ₁ is CH ₃ , Cobalt protoporphyrin (CoPP), an HO-1 inducer, increased HO-1 expression at 20 μM, which was used as a positive control. The results are shown in Figure 7 AD. Compound 1, wherein R ₁ is CH ₃ induced HO-1 expression in a dose- ( FIGS. 7A and B) and time-dependent manner ( FIGS. 7E and F).

In addition, the effect of compound 1 in which R ₁ is CH ₃ on nuclear translocation of Nrf2 was tested. As shown in Figures 7G and H, compound 1, wherein R ₁ is CH ₃ , significantly promoted the translocation of Nrf2 from the cytoplasm to the nucleus. From these results, it can be seen that compound 1 in which R ₁ is CH ₃ can promote the expression of HO-1 and regulate the Nrf2/HO-1 pathway.

Example 9: Compound 1 (R) in TNF-α/IFN-γ Stimulated HaCaT Cells _One =CH ₃ ) effect

Based on the result that Compound 1, wherein R ₁ is CH ₃ , can induce the expression of HO-1, we tested whether the anti-inflammatory effect of Compound 1 (R ₁ =CH ₃ ) is related to HO-1. Tin protoporphyrin IX (Snpp), a selective HO-1 inhibitor, was used, cells were treated with or without Snpp (40 μM) for 3 hours, and treated with or without Compound 1 (60 μM) in which R ₁ is CH ₃ , then 24 treated with TNF-α/IFN-γ for a period of time. Levels of IL-6, IL-8 and RANTES were determined by ELISA. As shown in FIGS. 8A-C , when compound 1 in which R ₁ is CH ₃ was treated, secretion of IL-6, IL-8, and RANTES induced by TNF-α/IFN-γ was significantly reduced. However, compared to the TNF-α/IFN-γ + compound 1 group, the secretion of IL-6, IL-8 and RANTES was reversed by the compound 1+Snpp group. Also, NF-κB binding activity showed similar results. SnPP treatment alone or TNF-α/IFN-γ+SnPP group did not affect cell viability, IL-6, IL-8, RANTES, and NF-κB binding activity. From these results, it can be seen that the anti-inflammatory effect of Compound 1 in which R ₁ is CH ₃ is mediated by HO-1 expression.

Example 10: Materials and Methods

Example 10-1: General Experimental Procedure

Optical rotations were recorded using a Jasco P-2000 digital polarimeter (JASCO Corp., Tokyo, Japan). Nuclear magnetic resonance (NMR) spectra (1D and 2D) were recorded in DMSO- d ₆ ( _δH / _δC = 2.50/39.52) using a JEOL JNM ECP-400 spectrometer (JEOL Ltd., Tokyo, Japan); Chemical shifts were referenced relative to residual solvent peaks. The binuclear multiple quantum coherence (HMQC) and binuclear multiple coupling correlation (HMBC) experiments were optimized for ¹ J _CH = 140 Hz and ⁿ J _CH = 8 Hz, respectively. HRESIMS data were obtained using an AB Sciex Triple TOF 4600 instrument (AB Sciex Pte. Ltd., Framingham, MA, USA). Thin layer chromatography (TLC) was performed on Kieselgel 60 F ₂₅₄ (1.05715; Merck, Darmstadt, Germany) or RP-18 F _254s (Merck) plates. After spraying 10% sulfuric acid aqueous solution (H ₂ SO ₄ ) on the plate, the spot was visualized by heating. Column chromatography was performed on silica gel (Kieselgel 60, 70-230 mesh and 230-400 mesh, Merck) and YMC octadecyl-functionalized silica gel (C ₁₈ ). High-performance liquid chromatography (HPLC) was performed on a preparative C ₁₈ column (10 Х 250 mm; 5 μm particle size) at a flow rate of 5 mL/min and compounds were detected by ultraviolet (UV) absorption at 210 and 254 nm.

Example 10-2: Fungal substances and fermentation

Fungal strain SF-7343 was isolated in January 2017 from moss collected from Marian Cove (62°13′16.5''S, 58°46′29.6''W) on King George Island, Antarctica. 1 g of the sample was ground in a mortar and pestle and mixed with sterile seawater (10 mL). An aliquot (0.1 mL) of the sample was processed using the spread plate method in PDA medium containing seawater and incubated at 25 °C for 14 days. After passage of the isolate several times, the final pure culture was selected and stored at -70 °C. Fungal strain SF-7343 was identified based on ITS gene sequencing. A GenBank search using the ITS gene of SF-7343 (GenBank accession number MK785420) identified Pleosporeles sp. Di61-4 (KC514877), Pleosporeles sp. AM282-P9T2T (KT264548) and Lophodermium pini-excelsae (EU520183) were the closest matches, exhibiting 100%, 99.58% and 99.37% sequence identity, respectively. The fungal strain SF-7343 was therefore characterized as Pleosporeles sp.

Example 10-3: Extraction and Separation of Metabolites

The fungal strain Pleosporeles sp. SF-7343 was cultured in 20 Fernbach flasks each containing 300 mL of PDB medium, 75 g of vermiculite and 3% NaCl. Flasks were individually inoculated with 5 mL of seed cultures of fungal strains and incubated at 25 °C for 14 days. Direct extraction of the cultured fungi with EtOAc (20 L) provided an organic phase which was then evaporated in vacuo to give a residue of SF-7343V (3.91 g). The crude extract was fractionated using RP C ₁₈ flash column chromatography eluting with a stepwise gradient of 20, 40, 60, 80 and 100% (v/v) MeOH in H ₂ O (500 mL each) , 6 fractions SF-7343V-1 to SF-7343V-6 were obtained. Compound 1 (9.0 mg) where R ₁ is H was fractionated by C ₁₈ preparative HPLC with elution with a gradient solvent system of 30-100% MeOH in H ₂ O containing 0.01% acid over 40 min. SF-7343V- 3 was purified from 30 mg (t _R = 27 min). Then, fraction SF-7343V-4 (1.6249 g) was applied to a silica gel column (32 x 3 cm) eluted with CH ₂ Cl ₂ -MeOH (100:1-0:100) to obtain 11 sub-fractions (SF- 7343V-4.1 to SF-7343V-4.11) were obtained. Of these sub-fractions, sub-fraction SF7343-4.8 (280.8 mg) was further separated with a silica gel column (20 x 2 cm) eluted with CH ₂ Cl ₂ -MeOH (125:1) to obtain sub-fractions SF7343-4.8.1 to SF7343- 4.8.5 was obtained. Sub-fraction SF7343-4.8.5 (49.5 mg) was subjected to C ₁₈ preparative HPLC (50-75% methanol in H ₂ O over 30 min) to give compound 3 (3.2 mg, t _R = 18.2 min). Finally, sub-fraction SF-7343V-4.9 (45.0 mg) was purified by C ₁₈ preparative HPLC (eluting with a gradient solvent system of 50-100% MeOH in H ₂ O over 30 min) to obtain compounds where R ₁ is CH ₃ 1 (4.2 mg, t _R = 19 min) and compound 2 (6.4 mg, t _R = 22 min) were obtained.

Compound 1, wherein R ₁ is CH ₃ , has the following characteristics:

Brown amorphous powder; HR-ESI-MS m/z : 327.0848 [M + Na] ⁺ (calcd. for [C ₁₆ H ₁₆ O ₆ Na] ⁺ , 327.0839). ¹ H NMR (DMSO- d ₆ , 400 MHz): 10.22 (s, O H -3), 8.73 (s, O H -11), 8.70 (s, O H -10), 6.55 (s, H -12 ), 6.42 (d, J = 2.4 Hz, H-4), 6.40 (s, H-9), 6.13 (d, J = 2.4 Hz, H-6), 3.72 (s, OCH ₃ -5), 3.41 (s, OCH 3 _-1 ), 1.87 (s, H -14); ¹³ C NMR (DMSO- d ₆ , 100 MHz): 168.4 (C-1), 160.8 (C-5), 157.4 (C-3), 144.2 (C-10), 143.0 (C-7), 142.2 ( C-11), 130.9 (C-8), 125.2 (C-13), 116.9 (C-12), 116.1 (C-9), 113.0 (C-2), 107.3 (C-6), 99.7 (C -4), 55.2 ( OCH ₃ -5), 51.4 ( OCH ₃ -1), 18.9 (C-14).

Compound 1, wherein R ₁ is H, has the following characteristics:

Yellowish powder; HR-ESI-MS m/z : 291.0878 [M + H] ⁺ (calcd. for [C ₁₅ H ₁₅ O ₆ ] ⁺ , 291.0863). ¹ H NMR (DMSO- d ₆ , 400 MHz): 8.67 (brs, O H -11), 8.63 (brs, O H -10), 6.54, (s, H -12), 6.43 (d, J = 2.7 Hz, H-4), 6.42 (s, H-9), 6.10 (d, J = 2.7 Hz, H-6), 3.76 (s, OCH _3-5 ), 1.86 (s, H-14); ¹³ C NMR (DMSO- d ₆ , 100 MHz): 171.6 (C-1), 162.0 (C-5), 161.5 (C-3), 145.0 (C-7), 144.0 (C-10), 142.2 ( C-11), 132.4 (C-8), 125.0 (C-13), 116.7 (C-12), 116.0 (C-9), 108.9 (C-6), 108.8 (C-2), 99.6 (C -4), 55.3 ( OCH ₃ -5), 18.9 (C-14).

Compound 2 has the following characteristics:

White powder; HR-ESI-MS m/z : 259.0606 [M + H] ⁺ (calcd. for [C ₁₄ H ₁₁ O ₅ ] ⁺ , 259.0601); 281.0428 [M + Na] ⁺ (Calcd. for [C ₁₄ H ₁₀ O ₅ Na] ⁺ , 281.0420). ¹ H NMR (DMSO- d ₆ , 400 MHz): 7.22 (d, J = 2.0 Hz, H-6), 6.70 (d, J = 2.7 Hz, H-12), 6.62 (d, J = 2.7 Hz, H-10), 6.35 (d, J = 2.0 Hz, H-4), 2.68 (s, H-14); ¹³ C NMR (DMSO- d ₆ , 100 MHz): 165.6 (C-5), 164.7 (C-1), 164.1 (C-3), 158.4 (C-11), 152.6 (C-9), 138.3 ( C-13), 138.1 (C-7), 117.5 (C-12), 109.0 (C-8), 104.4 (C-6), 101.6 (C-10), 100.9 (C-4), 97.2 (C -2), 25.2 (C-14).

Compound 3 has the following characteristics:

Brown amorphous powder; [ α ] ²⁵ _D −25.5 ( c 0.22, MeOH). HR-ESI-MS m/z : 293.1028 [M + H] ⁺ (calcd. for [C ₁₅ H ₁₇ O ₆ ] ⁺ , 293.1020); 315.0852 [M + Na] ⁺ (Calcd. for [C ₁₅ H ₁₆ O ₆ Na] ⁺ , 315.0839). ¹ H NMR (DMSO- d ₆ , 400 MHz): 11.28 (s, O H -3), 6.75 (d, J = 2.3 Hz, H-6), 6.51 (d, J = 2.3 Hz, H-4) , 6.30 (d, J = 3.2 Hz, H-6'), 5.31 (d, J = 5.6 Hz, O H -5'), 5.13 (brs, O H -4'), 3.95 (m, H-5 '), 3.86 (s, OC H ₃ -5), 3.70 (m, H -4'), 2.26 (dd, J = 3.2, 14.0 Hz, H _e -3'), 1.95 (dd, J = 7.2, 14.0 Hz, H _a -3'), 1.47 (s, H -7').

Example 10-4: Cell culture and reagents

HaCaT cells were donated by Professor Hyeon-Sook Jeong of Chosun University (Gwangju, Korea), and the cells were cultured in DMEM containing 10% FBS (Gibco, NY, USA) and tin protoporphyrin (Snpp; Calbiochem, Merck, Darmstadt, Germany). did ELISA kits for recombinant human TNF-α and IFN-γ, IL-8, IL-6 and RANTES were purchased from Bio-Legend (San Diego, CA, USA). Antibodies against ICAM-1, FLG, IVL, actin, PCNA, and HRP-binding anti-mouse and anti-rabbit IgG were purchased from Santa Cruz Bio-technology (Santa Cruz, CA, USA). Antibodies against p-IκBα, IκBα, p65, stat1, p-stat1, stat3, p-stat3, HO-1 and Nrf2 were purchased from Cell Signaling Technology (Danvers, MA, USA). Other chemical reagents were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA).

Example 10-5: MTT assay

To determine cell viability, cells were maintained at a density of 2 x 104 cells in 48 well plates, then compound 1 where R ₁ is CH ₃ (20-80 μM), compound 1 where R ₁ is H (5-40 μM) , treated with compound 2 (12.5-20 μM) and compound 3 (10-80 μM). After culturing for 24 hours, the cell culture medium was removed from each well and replaced with 500 μL of fresh medium in each well. Cells were incubated with 0.5 mg/mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) for 1 hour, and the formed formazan was incubated with dimethyl sulfoxide. (DMSO). The absorbance of dissolved formazan was measured at a wavelength of 540 nm using an ELISA microplate reader (Molecular Devices, San Jose, CA, USA).

Example 10-6: Measurement of cytokines and chemokines

HaCaT cells (5.0 Х 10 ⁶ cells/mL) were seeded in a 12-well plate. For ELISA assays, cells were pre-treated with compounds for 3 hours and then co-treated for 24 hours by stimulation with TNF-α/IFN-γ (5 ng/mL each). Cell culture supernatants were then collected and IL-6, IL-8, RANTES and MDC levels assessed using ELISA kits according to the manufacturer's instructions. For the experiment of Compound 1 in which R ₁ is CH ₃ using Snpp, HaCaT cells were first treated with Snpp (40 μM) for 2 hours, and then Compound 1 in which R ₁ is CH ₃ was added to the culture medium for another 3 hours. Finally, HaCaT cells were stimulated with TNF-α/IFN-γ for 24 hours. Thereafter, the cell culture supernatant was collected and IL-6, IL-8, and MDC were detected using an ELISA kit.

Example 10-7: Extraction of total protein, nuclear and cytoplasmic proteins

For total protein, cells were scraped, harvested, and lysed with RIPA buffer (Thermo Fisher Scientific, MA, USA). Nuclear and cytoplasmic protein fractions were extracted using a nuclear extraction kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer's instructions. All proteins were stored at -80 °C until use. HaCaT cells were pretreated with indicated concentrations of Compound 1 and Compound 2 where R ₁ is CH ₃ . For western blot analysis of ICAM-1, FLG and IVL, cells were induced with TNF-α/IFN-γ (5 ng/mL each) for 24 hours. For analysis of p-IκBα, IκBα, p65, stat1, p-stat1, stat3, and p-stat3, cells were induced with TNF-α/IFN-γ for 15 minutes.

Example 10-8: Western blot analysis

Proteins were electrophoretically separated on SDS-PAGE gels and transferred to nitrocellulose membranes. Membranes were blocked with 5% skim milk (dissolved in TBST) for 45-60 minutes at room temperature. It was then incubated overnight at 4 °C with primary antibody (1:1000 dilution). After washing three times with TBST, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (1:5000 dilution) for 1 hour at room temperature. After washing with TBST, specific proteins were detected using the ECL solution. Membranes were analyzed using ImageJ software (National Institutes of Health, Rockville, MD, USA).

Example 10-9: NF-κB DNA binding assay

For the experiment of Compound 1 in which R ₁ is CH ₃ using Snpp, HaCaT cells were first treated with Snpp (40 μM) for 2 hours, and then Compound 1 in which R ₁ is CH ₃ was added to the culture medium for another 3 hours. Finally, HaCaT cells were stimulated with TNF-α/IFN-γ. Then, the cells were harvested, and the nuclear protein fraction was extracted using a nuclear extraction kit.

NF-κB p65-DNA binding was detected using the NF-κB p65 transcription factor assay kit (10007889, Cayman Chemical). Nuclear fractionation was used to quantify relative nuclear NF-κB p65-DNA binding activity. This was evaluated according to the manufacturer's instructions.

Examples 10-10: statistical analysis

The results of each group were expressed as mean ± standard deviation (SD), and after performing one-way analysis of variance (ANOVA) using GraphPad Software (San Diego, CA, USA), Duncan's multiple comparison test was performed to determine significance. tested. Statistical significance was ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. control group. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. TNF-α/IFN-γ-treated group. ^Δp < 0.05, ^ΔΔp < 0.01 , ^ΔΔΔ p < 0.001 vs. The compound 1 +TNF-α/IFN-γ-treated group in which R ₁ is CH ₃ was set.

As above, specific parts of the present invention have been described in detail, and it will be clear to those skilled in the art that these specific descriptions are merely preferred embodiments, and the scope of the present invention is not limited thereby. will be. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Korea Research Institute of Bioscience and Biotechnology KCTC14724BP 20211005

Claims (8)

A pharmaceutical composition for preventing or treating inflammatory diseases comprising at least one compound selected from the group consisting of Formulas 1 to 3 below:
[Formula 1]

[Formula 2]

[Formula 3]

Here, the R ₁ to R ₆ are each independently H, straight-chain or branched-chain C _1-10 alkyl, C _1-10 alkoxy, C _3-10 cycloalkyl, C _2-10 alkenyl or C _6-10 Aryl.
The pharmaceutical composition according to claim 1, wherein R ₁ to R ₆ are each independently H, CH ₃ or CH ₂ CH ₃ .
The method of claim 1, wherein the compound is an Antarctic fungus Pleosporeles sp. A pharmaceutical composition characterized in that it is isolated from SF-7343 (KCTC 14724BP).
The method of claim 1, wherein the inflammatory disease is arthritis, rhinitis, hepatitis, keratitis, gastritis, enteritis, nephritis, bronchitis, pleurisy, peritonitis, spondylitis, pancreatitis, inflammatory pain, urethritis, cystitis, burn inflammation, dermatitis, periodontitis, gingivitis and A pharmaceutical composition, characterized in that selected from the group consisting of degenerative neuroinflammation.
The pharmaceutical composition according to claim 1, characterized in that it has one or more of the following properties:
1) inhibition of secretion of IL-6, IL-8, MDC (macrophage-derived chemokine) and RANTES;
2) inhibition of expression of ICAM-1;
3) inhibition of expression of filaggrin (FLG) and involcurin (IVL);
4) inhibition of activation of NF-κB, p-STAT1 and p-STAT3; and
5) Promotion of HO-1 expression and Nrf2 translocation.
The pharmaceutical composition according to claim 1, further comprising a pharmaceutically acceptable carrier, excipient or diluent.
Food for improving inflammation containing at least one compound selected from the group consisting of the following formulas 1 to 3:
[Formula 1]

[Formula 2]

[Formula 3]

Here, the R ₁ to R ₆ are each independently H, straight-chain or branched-chain C _1-10 alkyl, C _1-10 alkoxy, C _3-10 cycloalkyl, C _2-10 alkenyl or C _6-10 Aryl.
The food according to claim 7, wherein R ₁ to R ₆ are each independently H, CH ₃ or CH ₂ CH ₃ .

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