Anti-inflammatory composition containing 2,5,6-trimethoxy-p-terphenyl as effective component

Abstract

The present invention relates to an anti-inflammatory composition containing 2,5,6-trimethoxy-p-terphenyl as an active ingredient. Since TP1 (2,5,6-trimethoxy-p-terphenyl), which is the active ingredient of the present invention, has excellent effects in reducing NO and ROS increased by inflammatory response, inhibiting gene expression of IL-1β and IL-6, inhibiting gene expression and protein expression of iNOS and COX-2, reducing phosphorylation of AKT and NF-κB, and alleviating inflammatory symptoms in an animal model of sepsis induction, the composition of the present invention containing TP1 as an active ingredient can be usefully used as an anti-inflammatory composition.

Description

Technology Field

[0001] The present invention relates to an anti-inflammatory composition containing 2,5,6-trimethoxy-p-terphenyl as an active ingredient. Background Technology

[0002] Inflammation is a normal biological response that occurs in response to harmful stimuli, such as physical injury or infection by pathogens. As a type of defense mechanism, it is a protective reaction involving immune cells, blood vessels, and inflammatory mediators. The purpose of this inflammatory response is to suppress cell damage in the early stages, neutralize or eliminate pathogens, and simultaneously regenerate damaged tissues to restore their normal structure and function.

[0003] However, most diseases accompanied by inflammation can reduce the quality of life by causing symptoms such as heat, swelling, pain, and itching. In addition, in addition to acute inflammation caused by infection or physicochemical damage, chronic inflammation can persist due to lifestyle habits such as stress, smoking, and drinking, as well as conditions like arthritis, asthma, autoimmune diseases, and heart disease.

[0004] In cases of acute inflammation, the immune system is activated as immune cells, primarily macrophages, dendritic cells, histiocytes, and Kupffer cells, recognize stimuli. Pathogen-associated molecular types (PAMPs) and damage-associated molecular types (DAMPs) are present on the surface of stimulated cells; when tissue is damaged, these immune cells recognize these molecules, initiating signaling pathways via transcription factors such as NF-κB. Consequently, pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1 (IL-1), and interleukin-8 (IL-8) are secreted, followed by the release of inflammatory mediators that induce inflammatory symptoms. In particular, inflammatory factors such as nitric oxide (NO) and prostaglandin E2 (PGE2) are produced by macrophages; when the inflammatory response is excessive, large amounts of iNOS are generated in cells through this mechanism, followed by an increase in NO. Such excessive production of NO can lead to problems such as DNA structural damage, nerve damage, edema, and epithelial cell carcinoma.

[0005] Chronic inflammation is characterized by the gradual influence of inflammatory cells (e.g., monocytes) at the site of the reaction, and by the continuous occurrence of partial recovery and re-injury in the inflamed tissue. Because chronic inflammation sometimes persists for a long period without a recovery response following tissue damage, it can cause permanent damage.

[0006] Furthermore, in the presence of an inflammatory response, free radicals are generated along with such inflammation-related factors. While these free radicals generally participate in maintaining homeostasis, such as cell differentiation, growth, and survival, reactive oxygen species (ROS) are continuously produced through the oxidation and reduction processes of oxygen during respiration and immune responses. Because of their reactivity, they can have harmful effects on the body, so the body's antioxidant processes are designed to eliminate them. However, if this balance between generation and elimination is disrupted, oxidative stress occurs, which can lead to problems such as inflammation, aging, tissue damage, and cancer.

[0007] For the reasons mentioned above, despite the positive effects of inflammation, anti-inflammatory drugs are sometimes required; however, since most anti-inflammatory agents are synthetic compounds, efforts are needed to treat inflammation using naturally derived substances that are safer for the human body.

[0008] Meanwhile, sepsis is one of the most fatal diseases worldwide and is an acute inflammatory disease caused by an uncontrolled inflammatory response.

[0009] Although research on sepsis treatment is ongoing, there is currently no specific cure, and the management of sepsis patients relies primarily on antibiotics, intravenous fluids, and vasoactive agents. Ceftriaxone and vancomycin are commonly used antibiotics for sepsis, but they are known to cause numerous side effects, including urolithiasis, nephrolithiasis, and maculopapular or erythematous rashes. Therefore, due to the various side effects associated with sepsis treatment, there is a need to develop new low-toxicity anti-inflammatory agents for the treatment of sepsis.

[0010] Meanwhile, regarding prior art related to anti-inflammatory applications, Korean Registered Patent No. 2566485 discloses an anti-inflammatory composition containing Sargassum horneri extract as an active ingredient, Korean Registered Patent No. 2465346 discloses an anti-inflammatory composition containing a complex extract of Aster tataricus and Cudrania tricuspidata as an active ingredient, and Korean Registered Patent No. 2408812 discloses an anti-inflammatory composition containing Smilax china leaf extract, but there is no disclosure regarding an anti-inflammatory composition containing 2,5,6-trimethoxy-p-terphenyl (hereinafter TP1) of the present invention as an active ingredient. The problem to be solved

[0011] The present invention was derived from the above requirements and provides an anti-inflammatory composition containing TP1 (2,5,6-trimethoxy-p-terphenyl) as an active ingredient, and the invention was completed by confirming that TP1 inhibits an inflammatory response induced by LPS, alleviates symptoms in a sepsis-induced animal model, and has an anti-inflammatory effect. means of solving the problem

[0012] To solve the above problem, the present invention provides an anti-inflammatory pharmaceutical composition containing 2,5,6-trimethoxy-p-terphenyl or a pharmaceutically acceptable salt thereof as an active ingredient.

[0013] In addition, the present invention provides a health functional food composition for anti-inflammatory purposes containing 2,5,6-trimethoxy-p-terphenyl or a food-grade acceptable salt thereof as an active ingredient.

[0014] In addition, the present invention provides an anti-inflammatory cosmetic composition containing 2,5,6-trimethoxy-p-terphenyl or a cosmetically acceptable salt thereof as an active ingredient.

[0015] In addition, the present invention provides an anti-inflammatory quasi-drug containing 2,5,6-trimethoxy-p-terphenyl or a pharmaceutically acceptable salt thereof as an active ingredient.

[0016] In addition, the present invention provides a veterinary composition for anti-inflammatory purposes containing 2,5,6-trimethoxy-p-terphenyl or a pharmaceutically acceptable salt thereof as an active ingredient.

[0017] In addition, the present invention provides an anti-inflammatory feed additive containing 2,5,6-trimethoxy-p-terphenyl or a food-grade acceptable salt thereof as an active ingredient. Effects of the invention

[0018] The present invention relates to an anti-inflammatory composition containing 2,5,6-trimethoxy-p-terphenyl as an active ingredient. Since TP1 (2,5,6-trimethoxy-p-terphenyl), which is the active ingredient of the present invention, has excellent effects in reducing NO and ROS increased by inflammatory response, inhibiting gene expression of IL-1β and IL-6, inhibiting gene expression and protein expression of iNOS and COX-2, reducing phosphorylation of AKT and NF-κB, and alleviating inflammatory symptoms in an animal model of sepsis induction, the composition of the present invention containing TP1 as an active ingredient can be usefully used as an anti-inflammatory composition. Brief explanation of the drawing

[0019] Figure 1 shows the results of confirming changes in cell viability (A) and cell morphology (B) caused by TP1 treatment in LPS-treated Raw264.7 cells. Figure 2 shows the results of confirming the change in NO (Nitric oxide) production rate (A) and the change in ROS (Reactive oxygen species) production rate (B, C) caused by TP1 treatment in LPS-treated Raw264.7 cells. Figure 3 shows the results of confirming changes in mRNA levels of pro-inflammatory mediators (iNOS, COX-2, TNF-α, IL-6, and IL-1β) by TP1 treatment in LPS-treated Raw264.7 cells. * indicates that the mRNA levels of pro-inflammatory mediators in the LPS and TP1 treatment groups were statistically significantly reduced compared to the LPS-alone treatment group, and p<0.05. Figure 4 shows the results of confirming changes in the expression of pro-inflammatory enzymes (iNOS and COX-2) caused by TP1 treatment in LPS-treated Raw264.7 cells. Figure 5 shows the results of confirming the change in phosphorylation of AKT / NF-κB signaling proteins (A) and the change in intracellular localization of p65 (B) caused by TP1 treatment in LPS-treated Raw264.7 cells. Figure 6 shows the results of confirming the degree of phosphorylation of MAPK signaling proteins (ERK) by TP1 treatment in LPS-treated Raw264.7 cells. Figure 7 shows the experimental flowchart (A) for confirming the improvement effect of TP1 treatment in a sepsis-induced animal model, the results of quantifying the degree of pulmonary edema as the WET / Dry ratio of lung tissue (B), and the results of confirming the IL-1β mRNA level in spleen tissue (C). # indicates that the WET / Dry ratio of the LPS-alone treatment group was statistically significantly increased compared to the LPS-untreated group, and p<0.05. * indicates that the WET / Dry ratio or IL-1β mRNA expression of the LPS and TP1 treatment group was significantly decreased compared to the LPS-alone treatment group, and p<0.05. Specific details for implementing the invention

[0020] To achieve the objective of the present invention, the present invention provides a pharmaceutical composition for anti-inflammatory purposes containing 2,5,6-trimethoxy-p-terphenyl or a pharmaceutically acceptable salt thereof as an active ingredient.

[0021] The above 2,5,6-trimethoxy-p-terphenyl has the structure of Chemical Formula 1 below.

[0022]

[0023] The above 2,5,6-trimethoxy-p-terphenyl is stereocaulon alpinum ( Stereocaulon alpinum It is preferable to isolate it from ), but it is not particularly limited thereto, and it may be isolated from natural products other than stereocaulon alpinum, or synthesized by organic synthesis methods.

[0024] In one embodiment of the present invention, the anti-inflammatory may be an anti-inflammatory for sepsis, but is not limited thereto.

[0025] The composition of the present invention is preferably prepared in any one of the formulations selected from capsules, powders, granules, tablets, suspensions, emulsions, syrups, and aerosols, but is not limited thereto.

[0026] The composition of the present invention may further include pharmaceutically acceptable carriers, excipients, or diluents in addition to the active ingredient, and may be in various oral or parenteral formulations. When formulating, it is prepared using commonly used fillers, extenders, binders, wetting agents, disintegrants, surfactants, and other diluents or excipients. Solid formulations for oral administration include capsules, powders, granules, tablets, pills, etc., and these solid formulations are prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., with one or more compounds. In addition, lubricants such as magnesium stearate and talc are also used in addition to simple excipients. Liquid formulations for oral administration include suspensions, emulsions, syrups, and aerosols; in addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients, such as humectants, sweeteners, flavorings, and preservatives. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspension solvents. Witepsol, Macrogol, Tween 61, cocoa dough, laurin dough, and glycerogelatin may be used as bases for suppositories. For parenteral administration, it is preferable to select a method of administration via topical application or intraperitoneal, rectal, intravenous, intramuscular, subcutaneous, intrauterine dura mater, or intracerebrovascular injection.

[0027] 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 level of the effective amount may be determined based on factors including the type and severity of the patient's disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects by taking all of the above-mentioned factors into account, and this can be easily determined by a person skilled in the art.

[0028] The dosage of the composition of the present invention varies depending on the patient's body weight, age, gender, health status, diet, time of administration, method of administration, excretion rate, and severity of the disease. The composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modulators.

[0029] In addition, the present invention provides a health functional food composition for anti-inflammatory purposes containing 2,5,6-trimethoxy-p-terphenyl or a food-grade acceptable salt thereof as an active ingredient.

[0030] The above composition is preferably prepared in any one formulation selected from powder, granules, pills, tablets, capsules, candy, syrup, and beverage, but is not limited thereto.

[0031] When the health functional food composition of the present invention is used as a food additive, the active ingredient may be added as is or used together with other foods or food ingredients, and may be used appropriately according to conventional methods. The amount of the active ingredient can be appropriately determined according to its purpose of use (prevention, health, or therapeutic treatment). Generally, when manufacturing food or beverages, the composition of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, relative to the raw materials. However, in the case of long-term consumption for the purpose of health and hygiene or health control, the amount may be less than the above range, and since there are no issues regarding safety, the active ingredient may be used in an amount greater than the above range. There are no special restrictions on the types of food. Examples of foods to which the active ingredient can be added include meat, sausage, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and include all health functional foods in the conventional sense.

[0032] When the composition of the present invention is used as a health beverage, it may contain various flavoring agents or natural carbohydrates as additional ingredients, as in conventional beverages. The aforementioned natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclotensin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As sweeteners, natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame may be used. The proportion of the above natural carbohydrates is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 g of the composition of the present invention. The composition of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the composition of the present invention may contain fruit pulp for the production of natural fruit juices, fruit juice beverages, and vegetable beverages. These ingredients may be used independently or in combination. Although the proportion of these additives is not critical, the composition of the present invention is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight.

[0033] In addition, the present invention provides an anti-inflammatory cosmetic composition containing 2,5,6-trimethoxy-p-terphenyl or a cosmetically acceptable salt thereof as an active ingredient.

[0034] In a cosmetic composition according to one embodiment of the present invention, the cosmetic composition may be prepared in any one formulation selected from a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion, foundation, wax foundation, and spray, but is not limited thereto.

[0035] Cosmetic compositions consisting of each of these formulations may contain various bases and additives that are necessary and appropriate for the formulation of the formulation, and the types and amounts of these ingredients can be easily selected by those skilled in the art.

[0036] In the case where the formulation of the cosmetic composition of the present invention is a paste, cream, or gel, animal fiber, plant fiber, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as a carrier component.

[0037] When the formulation of the cosmetic composition of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, it may additionally include a propellant such as chlorofluorohydrocarbon, propane-butane, or dimethyl ether.

[0038] When the formulation of the cosmetic composition of the present invention is a solution or an emulsion, a solvent, a solvating agent, or an emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, or fatty acid ester of sorbitan.

[0039] In the case where the formulation of the cosmetic composition of the present invention is a suspension, liquid diluents such as water, ethanol, or propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracanth may be used as carrier components.

[0040] In the case where the formulation of the cosmetic composition of the present invention is a cleansing product containing a surfactant, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, acethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkylamidobetaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester, etc. may be used as a carrier component.

[0041] The cosmetic composition of the present invention may additionally contain excipients including fluorescent substances, fungicides, hydrotropism-inducing substances, moisturizers, fragrances, fragrance carriers, proteins, solubilizers, sugar derivatives, sunblocks, vitamins, plant extracts, etc.

[0042] In addition, the present invention provides an anti-inflammatory quasi-drug containing 2,5,6-trimethoxy-p-terphenyl or a pharmaceutically acceptable salt thereof as an active ingredient.

[0043] The above quasi-drugs may include external skin preparations and personal hygiene products. For example, they may be, but are not limited to, disinfectants, shower foams, mouthwash, wet wipes, detergent soaps, hand washes, or ointments.

[0044] When the above-described quasi-drug composition according to the present invention is used as a quasi-drug additive, the composition may be added as is or used together with other quasi-drugs or quasi-drug ingredients, and may be used appropriately according to conventional methods. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use.

[0045] The quasi-drug composition of the present invention may be prepared in the form of a general emulsion formulation and a solubilized formulation, for example. For example, it may have a formulation such as a lotion, cream, ointment, spray, oil gel, gel, oil, or aerosol, but is not limited to any other form as long as it exhibits the effects of the present invention.

[0046] In addition, the above quasi-drug composition may be used by appropriately incorporating oils, water, surfactants, humectants, lower alcohols having 1 to 4 carbon atoms, thickeners, chelating agents, colorants, preservatives, or fragrances, which are generally incorporated into quasi-drug compositions, into each formulation as needed.

[0047] In addition, the present invention provides a veterinary composition for anti-inflammatory purposes containing 2,5,6-trimethoxy-p-terphenyl or a pharmaceutically acceptable salt thereof as an active ingredient.

[0048] The veterinary composition of the present invention may further include suitable excipients and diluents according to conventional methods. Examples of excipients and diluents that may be included in the veterinary composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, cetanol, stearyl alcohol, liquid paraffin, sorbitan monostearate, polysorbate 60, methylparaben, propylparaben, and mineral oil.

[0049] The veterinary composition according to the present invention may further include fillers, anticoagulants, lubricants, wetting agents, spices, emulsifiers, preservatives, etc., and the veterinary composition according to the present invention may be formulated using methods well known in the art to provide rapid, sustained, or delayed release of the active ingredient after administration to an animal, and the formulation may be in the form of powders, granules, tablets, capsules, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, suppositories, sterile injectable solutions, sterile topical preparations, etc.

[0050] The effective amount of the veterinary composition according to the present invention can be appropriately selected according to the individual animal. It may be determined based on the severity of the disease or condition, the sensitivity to the active ingredient of the present invention according to the individual's age, weight, health status or gender, the route of administration, the duration of administration, factors including other compositions combined with or used simultaneously with the composition, and other factors well known in the physiological or veterinary field.

[0051] In addition, the present invention provides an anti-inflammatory feed additive containing 2,5,6-trimethoxy-p-terphenyl or a food-grade acceptable salt thereof as an active ingredient.

[0052] The feed additive of the present invention corresponds to an auxiliary feed under the Feed Management Act. In the present invention, the term "feed" may refer to any natural or artificial prescribed food, single meal, etc., or the components of said single meal, intended for or suitable for animals to eat, consume, and digest. The types of said feed are not particularly limited, and feeds commonly used in the relevant technical field may be used. Non-limiting examples of said feed include plant-based feeds such as grains, root vegetables, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, meal, or grain by-products; and animal-based feeds such as proteins, inorganic substances, oils and fats, minerals, single-cell proteins, zooplankton, or food waste. These may be used individually or in a mixture of two or more types.

[0054] The present invention will be described in more detail below using examples. These examples are intended solely to explain the present invention more specifically, and it is obvious to those skilled in the art that the scope of the present invention is not limited by them.

[0056] Materials and Methods

[0057] Reagent

[0058] TP1 was extracted and provided by the Korea Polar Research Institute and dissolved in DMSO (dimethyl sulfoxide).

[0059] DMEM (Dulbecco's modified Eagle's medium) was purchased from Hyclone Laboratories Inc. (Marlborough, MA, USA), and FBS (fetal bovine serum) was purchased from Corning (Corning, NY, USA). PSQ (Penicillin-streptomycin-glutamine) was purchased from Gibco (Waltham, MA, USA), and MTT, E. coli ( Escherichia coli LPS derived from O127:B8, N-(1-Naphthyl)ethylenediamine dihydrochloride, phosphoric acid, sulfanilic acid, and nitrite ion standard solutions were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).

[0060] DPBS (Dulbecco's phosphate-buffered saline) and HBSS (Hank's balanced salt solution) were purchased from WELGENE, Inc. (Gyeongsan, Korea). DCF-DA (2'7-Dicholrodihydrofluorecsein diacetate) was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). qRT-PCR (quantitative reverse transcription polymerase chain reaction) primers were purchased from Macrogen (Seoul, Korea).

[0061] The primary antibodies [iNOS (#13120), COX-2 (#12282), phospho-p65 (#13346), phospho-ERK (#4370) and β-actin (#8457)] were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA), and the goat anti-rabbit IgG (5220-0036) and goat anti-mouse IgG (5220-0341) antibodies were purchased from SeraCare Life Sciences, Inc. (Gaithersburg, MD, USA).

[0063] Cell culture

[0064] The mouse-derived Raw264.7 macrophage cell line was provided by Dr. Sung-Ho Ryu's laboratory (POSTECH, Korea). Cells were cultured in DMEM medium containing 10% (v / v) FBS and 1% (v / v) PSQ in a 5% CO2, 37°C incubator.

[0066] Cell viability (MTT assay)

[0067] 3×10 4 Canine cells were seeded into each well of a 48-well plate and stabilized for 24 hours. Then, they were pretreated with TP1 for 30 minutes, followed by treatment with 0.1 μg / mL of LPS, and cultured for 24 hours in a 5% CO2, 37°C incubator. Afterward, the culture medium was removed, and MTT solution (1 mg / mL) was added and reacted for 2 hours. After removing the MTT solution, 200 μl of DMSO was added to each well to dissolve the formazan crystals. Subsequently, the absorbance was measured at 570 nm using a microplate reader (Varioskan LUX Multimode Microplate Reader, Thermo Fisher Scientific Co.).

[0069] Measurement of Nitric Oxide Production

[0070] Griess assay was used to measure NO in the cell culture supernatant.

[0071] 6×10 4 Raw264.7 cells were seeded into each well of a 24-well plate and stabilized for 24 hours, then pretreated with TP1 (25 μM) for 30 minutes. Afterward, 0.1 μg / mL of LPS was added, and the cells were incubated in a 5% CO2, 37°C incubator for 24 hours. After incubation, the supernatant from each well was transferred to a 96-well plate, and Griess reagent with a 1:1 volume ratio of reagents A and B was added. The mixture was then reacted for 15 minutes, and the absorbance was measured at a wavelength of 550 nm using a microplate reader.

[0073] Measurement of Reactive Oxygen Species Production

[0074] ROS production in Raw264.7 cells was measured using DCF-DA analysis.

[0075] 6×10 4 100 Raw264.7 cells were seeded into each well of a 24-well plate and stabilized for 24 hours, then pretreated with TP1 (25 μM) for 30 minutes. Afterward, 0.1 μg / mL of LPS was added, followed by incubation in a 5% CO2, 37°C incubator for 24 hours. Subsequently, DCF-DA was added at 37°C for 30 minutes, and the cells were washed three times with HBSS before measuring fluorescence. The level of ROS production was detected using a fluorescence microscope (Zeiss, Jena, Germany) and analyzed using FACS (BD FACSVerse™, BD Bioscience).

[0077] qRT-PCR (quantitative reverse transcription polymerase chain reaction)

[0078] 3×10 510 Raw264.7 cells were seeded into each well of a 12-well plate and stabilized for 24 hours, after which they were pretreated with TP1 (25 μM) for 30 minutes. Subsequently, 0.1 μg / mL of LPS was added, and the cells were incubated in a 5% CO2, 37°C incubator for 24 hours. After 24 hours, the cells were washed three times with DPBS and harvested. Total RNA was then isolated using the TRIzol reagent, and cDNA synthesis was performed using the total RNA according to the protocol of the SimpliAmp Thermal Cycler (Applied Biosystems Co., Waltham, MA, USA). Finally, qRT-PCR was performed using the Power SYBR Green PCR mix Cycler (Applied Biosystems Co.) and the Step One Plus Real-time PCR System Cycler (Applied Biosystems Co.).

[0080] Western blot analysis

[0081] 6×10 5 After dispensing cells into a 60 mm culture dish and stabilizing them for 24 hours, they were treated with 25 μM TP1 for 30 minutes. Then, after treating with 0.1 μg / mL LPS, 100 μl of RIPA buffer (10 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA pH 8, 1% Triton X-100, 1% sodium deoxycholate, 30 mM NaF, 1.5 mM NaVO4, 1 mM PMSF, 1 mg / mL aprotinin, 1 mg / mL leupeptin, and 1 mg / mL pepstatin A) was added to each sample after 30 minutes, 2 hours, or 24 hours, and cell lysates were obtained by sonication three times for 5 seconds. The cell lysate was centrifuged at 4°C and 130,000 rpm for 10 minutes to obtain the supernatant, and the protein was quantified using Bradford assay (Abcam, Cambridge, UK).

[0082] Total proteins were separated by SDS-PAGE, transferred to a nitrocellulose (NC) membrane, and then blocked for 30 minutes using 5% (w / v) skim milk, 0.1% Tween-20, and 0.01% (w / v) NaN3 dissolved in TBS. Subsequently, the primary antibody was diluted in the same blocking solution and incubated overnight, after which the secondary antibody was applied to the membrane and reacted at room temperature for 1 hour. After the reaction, an ECL solution was applied to the membrane, and proteins were detected and bands visualized using Chemidoc (iBright™ CL1500, Invitrogen).

[0084] Immunocytochemistry

[0085] 5×10 5Raw264.7 cells were seeded into each well of a 6-well plate and stabilized for 24 hours, then pretreated with TP1 (25 μM) for 30 minutes. After that, 0.1 μg / mL of LPS was treated for 2 hours, then the cells were washed with DPBS, and fixed with 4% (v / v) paraformaldehyde (PFA, Sigma-Aldrich; Merck KGaA) for 20 minutes. Subsequently, the cells were incubated for 5 minutes with PBS containing 0.2% Triton X-100 and 0.1% citrate (Sigma-Aldrich; Merck KGaA), blocked with 2% BSA (Sigma-Aldrich; Merck KGaA) for 30 minutes, and then probed with rabbit anti-p65 NF-κB antibody (1:500; cat. no. 8242; Cell Signaling Technology, Inc.) for 2 hours. Afterward, the cells were washed with 2% BSA and incubated for 1 hour with the fluorescein goat anti-rabbit IgG(H+L) cross-linked secondary antibody Alexa Fluor 555 (4 µg / mL; cat. no. A-21428; Invitrogen Inc., Middlesex County, MA, USA). Afterward, the cells were washed with 2% BSA, and the nuclei were stained with 1 mg / mL DAPI solution for 5 minutes under light-free conditions. After 5 minutes, the fluorescence was visualized using a fluorescence microscope (Zeiss microscope, Carl Zeiss AC).

[0087] LPS-induced septic murine model

[0088] Animal experiments were conducted using male BALB / C mice (8 weeks old). The mice were purchased from Orient Bio (Seongnam, Korea) and managed in accordance with the approval guidelines of the Institutional Animal Care and Use Committee (IACUC) of the School of Applied Life Sciences, Gyeongsang National University.

[0089] For the experiment, mice were randomly divided into three groups (n=5), and 15 mg / kg TP1 (TP1) or PBS (control, LPS) was injected intraperitoneally 2 hours prior to LPS injection. Subsequently, 5 mg / kg of LPS (LPS) or PBS (control, TP1) was injected intraperitoneally, and after monitoring for 24 hours, the mice were sacrificed.

[0091] WET / DRY ratio

[0092] After extracting the lungs from the sacrificed animal model above, their weight was measured. Subsequently, the lung tissue was dried at 65°C for 24 hours.

[0093] The weight of the dry lungs was measured, and the ratio was calculated by dividing the weight of the wet lungs by the weight of the dry lungs.

[0095] Tissue mRNA isolation

[0096] After extracting the spleen of the sacrificed animal model, it was frozen at -80℃. Subsequently, the frozen spleen was crushed with a mortar and pestle using liquid nitrogen, and total RNA was isolated using the TRIzol reagent, followed by cDNA and qRT-PCR.

[0098] Example 1. Cell viability and cell morphology changes induced by TP1 treatment in LPS-treated Raw264.7 cells

[0099] To confirm the cytotoxicity of TP1, MTT analysis and observation of cell morphological changes were performed. As a result, as disclosed in Figure 1, it was confirmed that TP1 had no cytotoxicity up to a concentration of 25 μM and inhibited LPS-induced cell morphological changes.

[0101] Example 2. Changes in NO (Nitric oxide) and ROS (Reactive oxygen species) production rates by TP1 treatment in LPS-treated Raw264.7 cells

[0102] Increased ROS production can lead to endothelial dysfunction and tissue damage, and NO plays various roles in inflammatory responses, such as regulating leukocyte activation and tissue cytotoxicity, potentially causing vasodilation and edema. Therefore, the production of NO and ROS by TP1 treatment was confirmed.

[0103] As a result, as disclosed in Figure 2A, it was confirmed that the production of NO increased by LPS treatment decreased in a TP1 concentration-dependent manner.

[0104] In addition, the generation of ROS was confirmed using FACS and fluorescence microscopy, and as disclosed in Figures 2B and 2C, it was confirmed that TP1 reduces ROS production in a dose-dependent manner.

[0105] Through this, it was confirmed that TP1 can reduce NO and ROS production in LPS-induced Raw264.7 cells.

[0107] Example 3. Changes in mRNA levels of pro-inflammatory mediators (iNOS, COX-2, TNF-α, IL-6, and IL-1β) induced by TP1 treatment in LPS-treated Raw264.7 cells

[0108] We confirmed whether TP1 can regulate the expression of pro-inflammatory mediators at the mRNA level.

[0109] As a result, as disclosed in Figure 3, it was confirmed that the TP1 treatment group reduced the mRNA expression of iNOS, COX-2, TNF-α, IL-6, and IL-1β compared to the LPS-alone treatment group.

[0112] Example 4. Changes in the expression of pro-inflammatory enzymes (iNOS and COX-2) induced by TP1 treatment in LPS-treated Raw264.7 cells

[0113] In LPS-treated Raw264.7 cells, changes in the expression of pro-inflammatory enzymes (iNOS and COX-2) by TP1 treatment were confirmed, and as disclosed in Figure 4, the TP1-treated group significantly reduced the protein expression of iNOS and COX-2 compared to the LPS-alone treated group.

[0115] Example 5. Changes in phosphorylation of AKT / NF-κB signaling proteins and changes in the intracellular localization of p65 induced by TP1 treatment in LPS-treated Raw264.7 cells

[0116] NF-κB is a key transcription factor that regulates immune cells, including inflammatory T cells, and induces the expression of pro-inflammatory enzymes and cytokine mRNAs following LPS stimulation. Additionally, activated AKT induces the degradation of nuclear factor B inhibitor (IκB) protein, and p65 promotes nuclear translocation by enhancing the phosphorylation of NF-κB subunits. Therefore, we investigated whether TP1 could regulate the phosphorylation levels of AKT and p65 in LPS-induced Raw264.7 cells and examined the nuclear translocation of p65 through immunocytochemistry.

[0117] As a result, as disclosed in Fig. 5A, the phosphorylation of AKR and p65 in the TP1 treatment group was downregulated compared to the LPS alone treatment group, and as disclosed in Fig. 5B, the translocation of p65 to the nucleus increased by LPS treatment was inhibited by TP1 treatment.

[0119] Example 6. Degree of phosphorylation of MAPK signaling proteins by TP1 treatment in LPS-treated Raw264.7 cells

[0120] Since the MAPK signaling pathway plays a crucial role in initiating cellular inflammatory responses, including the production of pro-inflammatory cytokines in response to LPS stimulation, we examined the phosphorylation levels of ERK induced by TP1 treatment.

[0121] As a result, as disclosed in Figure 6, TP1 reduced the phosphorylation of ERK induced by LPS.

[0123] Example 7. Effect of TP1 treatment on sepsis improvement in a sepsis-induced animal model

[0124] LPS is a major cause of sepsis, stimulating the immune system and inducing severe inflammation. Therefore, to evaluate the anti-inflammatory effects of TP1 in vivo, an LPS-induced mouse sepsis model was established as disclosed in Figure 7A, and the effects of TP1 were confirmed.

[0125] As a result of measuring the degree of pulmonary edema as a WET / Dry ratio, it was confirmed that the WET / Dry ratio of the TP1 treatment group was reduced compared to the LPS-alone treatment group as disclosed in Fig. 7B, and that mRNA expression of the pro-inflammatory cytokine IL-1β was significantly reduced in the TP1 treatment group compared to the LPS-alone treatment group as disclosed in Fig. 7C.

[0126] Through this, it was confirmed that TP1 can alleviate sepsis symptoms such as pulmonary edema by downregulating the gene expression of pro-inflammatory cytokines in an LPS-induced sepsis animal model.

Claims

Claim 1 A pharmaceutical composition for anti-inflammatory purposes containing 2,5,6-trimethoxy-p-terphenyl or a pharmaceutically acceptable salt thereof as an active ingredient. Claim 2 In paragraph 1, the active ingredient is stereocaulon alpinum ( Stereocaulon alpinum A pharmaceutical composition for anti-inflammatory purposes characterized by being separated from ). Claim 3 A pharmaceutical composition for anti-inflammatory purposes, characterized in that, in claim 1, the anti-inflammatory agent is an anti-inflammatory agent for sepsis. Claim 4 An anti-inflammatory pharmaceutical composition according to claim 1, characterized in that the composition is prepared in any one formulation selected from capsules, powders, granules, tablets, suspensions, emulsions, syrups, and aerosols. Claim 5 A pharmaceutical composition for anti-inflammatory purposes according to claim 1, wherein the composition further comprises, in addition to the active ingredient, a pharmaceutically acceptable carrier, excipient, or diluent. Claim 6 An anti-inflammatory health functional food composition containing 2,5,6-trimethoxy-p-terphenyl or a food-grade acceptable salt thereof as an active ingredient. Claim 7 An anti-inflammatory health functional food composition according to claim 6, characterized in that the composition is manufactured in any one formulation selected from powder, granules, pills, tablets, capsules, candies, syrups, and beverages. Claim 8 An anti-inflammatory cosmetic composition containing 2,5,6-trimethoxy-p-terphenyl or a cosmetically acceptable salt thereof as an active ingredient. Claim 9 An anti-inflammatory quasi-drug containing 2,5,6-trimethoxy-p-terphenyl or a pharmaceutically acceptable salt thereof as an active ingredient. Claim 10 A veterinary composition for anti-inflammatory purposes containing 2,5,6-trimethoxy-p-terphenyl or a pharmaceutically acceptable salt thereof as an active ingredient. Claim 11 Anti-inflammatory feed additive containing 2,5,6-trimethoxy-p-terphenyl or a food-acceptable salt thereof as an active ingredient.

Images