Pharmaceutical composition for preventing or treating arthritis containing Usenamine A

Abstract

The present invention aims to provide a pharmaceutical composition for the prevention or treatment of arthritis that is effective in alleviating arthritis by inhibiting the expression of inflammatory cytokines involved in arthritis. Specifically, the composition may have an arthritis-alleviating effect by inhibiting the secretion of inflammatory cytokines such as IL-17, GM-CSF, and INF-gamma.

Description

Technology Field

[0001] The present invention relates to a pharmaceutical composition for the prevention or treatment of arthritis comprising Usenamine A as an active ingredient. Background Technology

[0002] Immunological diseases are conditions in which components of the mammalian immune system cause, mediate, or otherwise contribute to pathological conditions in mammals; in particular, inflammatory disorders are one of the most critical health issues worldwide. Inflammation is generally a localized protective response of body tissues against host invasion by external substances or harmful stimuli. Causes of inflammation can be infectious agents such as bacteria, viruses, and parasites; physical agents such as burns or radiation; chemicals such as toxins, drugs, or industrial agents; immunological responses such as allergies and autoimmune reactions; or conditions associated with oxidative stress.

[0003] Inflammation is characterized by pain, reddening, swelling, fever, and the ultimate loss of function in the infected area. These symptoms are the result of a series of complex interactions occurring among cells of the immune system. Cellular responses consequently generate an interaction network of various groups of inflammatory mediators. Types of inflammatory mediators include proteins, basic proteins, adhesion molecules (ICAM, VCAM), lipid mediators (eicosanoids, prostaglandins, leukotrienes, platelet-activating factors), and reactive oxygen species (hydroperoxide, superoxide anion O2-, nitric oxide). However, most of these also act as regulators of normal cellular activity. Consequently, a lack of inflammatory response leads to infection when the host is not properly controlled, and inflammatory diseases mediated by the overproduction of several of the aforementioned mediators occur.

[0004] Rheumatoid arthritis is an inflammatory autoimmune disease that manifests in multiple joints. In patients with arthritis, synovial tissue proliferates; simultaneously, macrophages, dendritic cells, and activated T and B lymphocytes migrate into the synovial tissue, while polymorphonuclear cells accumulate in the synovial fluid and on the cartilage surface, causing inflammation. This inflammation of the synovial tissue is known to be triggered by T lymphocytes that are reactive to unknown autoantigens. Nevertheless, T lymphocytes infiltrating most tissues do not display activation markers on their cell surfaces and rarely express cytokines.

[0005] In contrast, a large number of cytokines derived from macrophages are observed in the synovial tissue and synovial fluid associated with symptoms of rheumatoid arthritis. These cytokines include interleukin-1 (IL-1) and tumor necrosis factor-(TNF-α), which are known to promote the growth of synovial fibroblasts. These experimental results suggest a hypothesis that T lymphocytes play a crucial role in inducing inflammation in synovial tissue, and that subsequent inflammatory symptoms are maintained by cytokines derived from activated synovial cells.

[0006] Synovial tissue proliferation in arthritis forms pannus, a layer of connective tissue that occurs in the avascular cartilage, which irreversibly destroys the cartilage and bone tissue of the affected joint. This is caused by the action of matrix metalloproteinases (MMPs), known as cartilage tissue destruction factors, which destroy the cartilage tissue.

[0007] As described above, the ideal goal of treating arthritis, such as rheumatoid arthritis, is to protect and maintain the cartilage and bone tissue of the affected joint. To this end, research on various pharmaceutical compositions for improving bone inflammation and health functional foods containing them has been actively conducted, and recently, research has been progressing in the direction of extracting active ingredients effective for improving bone inflammation from natural substances. Prior art literature

[65535] Korean Registered Patent Publication No. 10-2883839 (Publication Date: March 31, 2021) The problem to be solved

[0008] The present invention aims to provide a pharmaceutical composition for the prevention or treatment of arthritis that is effective in alleviating arthritis by inhibiting the expression of inflammatory cytokines involved in arthritis. means of solving the problem

[0009] The present disclosure provides a composition for the prevention or treatment of arthritis comprising Usenamine A as an active ingredient.

[0010] The above Usenamine A may be extracted from Songra.

[0011] The above Usenamine A is obtained by the step of extracting dried pine needles using an organic solvent;

[0012] It may be extracted through a method comprising the steps of: dissolving the above extract in distilled water; fractionating the dissolved extract through chromatography; and obtaining Usenamine A from the fraction.

[0013] The above extraction step may include performing ultrasonic extraction one or more times.

[0014] The above composition may contain Usenamine A at a concentration of 0.1 to 50 μg / ml.

[0015] The above composition may inhibit one or more selected from INF-gamma, IL-17, and GM-CSF.

[0016] The above anti-inflammatory composition may be prepared in any one formulation selected from capsules, topical preparations, powders, granules, tablets, suspensions, emulsions, syrups, and aerosols.

[0017] The above anti-inflammatory composition may further include a pharmaceutically acceptable carrier, excipient, or diluent in addition to the active ingredient.

[0018] The present disclosure provides a health functional food composition for the prevention or improvement of arthritis comprising Usenamine A as an active ingredient.

[0019] The above composition may be prepared in any one formulation selected from powder, granules, pills, tablets, capsules, candy, syrup, and beverage. Effects of the invention

[0020] The pharmaceutical composition for the prevention or treatment of arthritis according to the present invention may have an arthritis-relieving effect by inhibiting the secretion of inflammatory cytokines such as IL-17, GM-CSF, and INF-gamma. Brief explanation of the drawing

[0021] Figure 1 shows the results of a cytotoxicity test for Usenamine A treatment. Figure 2 shows the expression rates of each inflammatory cytokine according to Usenamine A concentration for Usenamine A treatment. Figure 3 shows the change in the arthritis index over time for each sample treatment. Specific details for implementing the invention

[0022] The present invention will be described in detail below. Unless otherwise defined, terms used in this specification should be interpreted as generally understood by those skilled in the art. The drawings and embodiments of this specification are intended to enable those skilled in the art to easily understand and practice the present invention; therefore, details that may obscure the essence of the invention may be omitted from the drawings and embodiments, and the present invention is not limited to the drawings and embodiments.

[0023] The singular form used in this specification may be intended to include the plural form unless specifically indicated otherwise in the context.

[0024] Furthermore, the numerical range used in this invention includes lower and upper limits and all values ​​within the range, increments logically derived from the form and width of the defined range, all of the specified values, and all possible combinations of upper and lower limits of the numerical range defined in different forms. Unless otherwise specifically defined in the specification of this invention, values ​​outside the numerical range that may occur due to experimental error or rounding are also included in the defined numerical range.

[0025] In this specification, terms such as "include," "have," and "have" mean that the features or components described in the specification are present, and unless specifically limited, this does not preclude the possibility that one or more other features or components may be added.

[0026] The present disclosure provides a pharmaceutical composition for the prevention or treatment of arthritis comprising Usenamine A as an active ingredient.

[0027] A pharmaceutical composition for the prevention or treatment of arthritis according to one embodiment of the present disclosure may have anti-inflammatory activity, such as wound healing activity, and immune modulatory function, and specifically may have an effect of suppressing arthritis. The composition may have an effect of improving immune function by suppressing the expression of IL-17, GM-CSF, and INF-gamma, which are major causative factors of rheumatoid arthritis.

[0028] The above Usenamine A may be extracted from Songra.

[0029] Usnea diffracta is a lichen belonging to the genus Usnea in the family Pulmonaceae; it has very long stems and very short branches. It is found in the boreal forests (taiga) of Europe, Asia, and North America, and is used in traditional Korean medicine as a diuretic, antipyretic, and expectorant.

[0030] The above Usenamine A is obtained by the step of extracting dried pine needles using an organic solvent;

[0031] It may be extracted through a method comprising the steps of: dissolving the above extract in distilled water; fractionating the dissolved extract through chromatography; and obtaining Usenamine A from the fraction.

[0032] The above dried pine is pine ( Usnea diffracta) The process may involve freeze-drying, vacuum drying, hot air drying, spray drying, vacuum drying, foam drying, high-frequency drying, or infrared drying, and may include, but is not limited to, a process of crushing the dried pine needles.

[0033] The above organic solvent may be methanol, ethanol, isopropanol, butanol, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, dichloromethane, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, or a mixture thereof, and extraction may be performed at room temperature or under heating conditions where the active ingredients of the extract are not destroyed or are minimized.

[0034] The above extraction step may include performing ultrasonic extraction one or more times, and specifically, may include performing it three or more times.

[0035] The above pharmaceutical composition for the prevention or treatment of arthritis may contain Usenamine A at a concentration of 0.1 to 50 μg / ml, and specifically may contain it at a concentration of 5 to 30 μg / ml.

[0036] The above pharmaceutical composition for the prevention or treatment of arthritis may inhibit one or more selected from INF-gamma (Interferon gamma), IL-17 (interleukin-17), and GM-CSF (granulocyte-macrophage colony-stimulating factor).

[0037] Cytokines INF-gamma, IL-17, and GM-CSF act on fibroblasts, endothelial cells, and epithelial cells to influence various biological responses, primarily engaging in inflammatory and hematopoietic functions. In rheumatoid arthritis, IL-17 is produced by Th17 lymphocytes in the synovium and acts on synovial cells, monocytes, macrophages, and chondrocytes to release various cytokines, playing a major role in joint inflammation and destruction. INF-gamma and IL-17 are produced by CD4+ or CD8+ cells and have been identified as playing important roles in the onset and activation of arthritis. Furthermore, GM-CSF is a pro-inflammatory cytokine involved in the survival, differentiation, and activation of macrophages, dendritic cells, and neutrophils; GM-CSF cytokines and their receptors are overexpressed in synovial tissue or monocytes of patients with rheumatoid arthritis.

[0038] The above pharmaceutical composition for the prevention or treatment of arthritis may be prepared in any one of the formulations selected from capsules, topical preparations, powders, granules, tablets, suspensions, emulsions, syrups, and aerosols, and specifically may have the form of tablets.

[0039] The above pharmaceutical composition for the prevention or treatment of arthritis may be in a solid or liquid form, and the method of administration may be oral, parenteral, intranasal, oral, sublingual, airway spray, or rectal, but is not limited thereto, and according to the results of one embodiment of the present invention, it may have a form of administration via injection, etc. during parenteral administration.

[0040] The above pharmaceutical composition for the prevention or treatment of arthritis may further include a pharmaceutically acceptable carrier, excipient, or diluent in addition to the active ingredient. The carrier, excipient, and diluent may be various compounds or mixtures including 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, and mineral oil.

[0041] The present disclosure provides a health functional food composition for the prevention or improvement of arthritis comprising Usenamine A as an active ingredient.

[0042] The above-mentioned health functional food composition for preventing or improving arthritis may be manufactured in any one formulation selected from powder, granules, pills, tablets, capsules, candies, syrups, and beverages.

[0043] The above-mentioned health functional food composition is not particularly limited as long as it can be consumed to prevent or improve diseases. When the health functional food composition of the present invention is used as a food additive, it may be added as is or used together with other foods or food ingredients, and may be used appropriately according to conventional methods. The active ingredient may be used appropriately according to its intended use. Generally, when manufacturing food or beverages, the health functional food composition of the present disclosure is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less. However, in the case of long-term consumption for the purpose of health control, the above 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.

[0044] The above-mentioned health functional food composition includes ingredients that are typically added during food manufacturing, for example, proteins, carbohydrates, fats, nutrients, and flavoring agents. For example, when manufactured as a drink, natural carbohydrates or flavoring agents may be included as additional ingredients in addition to the active ingredients. The above-mentioned natural carbohydrates are preferably monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, etc.), oligosaccharides, polysaccharides (e.g., dextrin, cyclodextrin, etc.), or sugar alcohols (e.g., xylitol, sorbitol, erythritol, etc.). The above-mentioned flavoring agents may include natural flavoring agents (e.g., taumatin, stevia extract, etc.) and synthetic flavoring agents (e.g., saccharin, aspartame, etc.). In addition to the above health functional food composition, it may further contain various nutritional supplements, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc.

[0045] Hereinafter, a health functional food composition for the prevention or improvement of arthritis according to the present invention will be described in more detail through specific examples. However, the following examples are merely references for the detailed explanation of the present invention and the present invention is not limited thereto and may be implemented in various forms. Furthermore, the terms used in the description of the present invention are intended merely to effectively describe specific examples and are not intended to limit the present invention.

[0047] [Example 1] Extraction of Usenamine A

[0048] 1.5 kg by dry weight using 100% methanol as a solvent at room temperature Usnea diffractaUltrasonic extraction was performed three times. After extraction, the solvent was removed by vacuum to obtain approximately 285.7 g of methanol extract. The extract was then dissolved in distilled water and sequentially separated into ethyl acetate (EtOAc) and n-butyl alcohol to obtain two powder extracts. The ethyl acetate extract (125.4 g) was used with a silicate column chromatography to produce eight distinct fractions (E1-E8) using a gradient solvent with a mobile phase ratio of dichloromethane to methanol ranging from 100:1 to 1:100 (v / v). Fraction E3 (35.1 g) was then treated with a methanol gradient (10% to 100%) using reverse-phase C18-medium-pressure liquid chromatography (RP C18-MPLC) to produce 13 additional subfractions (M1-M13). Of these, fraction M12 was further separated using silica gel column chromatography with a hexane-ethyl acetate solvent system in a ratio of 10:1 to 1:1 to produce a total of five new subfractions (M14-M18). Finally, subfraction M17 was separated via pretreatment high-performance liquid chromatography (HPLC) using 70% acetonitrile as the mobile phase to obtain the final product, usenamine A.

[0050] [Evaluation Example 1] Cell stability evaluation

[0051] Peripheral blood mononuclear cells were isolated from the blood of a normal donor. Inflammation was induced with CD3 / 28 to stimulate the cells, and the inflammation-induced immune cells were treated with usenamine A extracted in Preparation Example 1 at concentrations of 0, 0.1, 1, 3, 5, 10, and 20 µg / ml, respectively. The results of comparing the cytotoxicity of each treatment group using the MTS assay are shown in Figure 1.

[0052] As shown in Figure 1, cell viability decreased slightly as the treatment dose increased, but it can be seen that Usenamine A treatment provides cell stability through the fact that it maintained a high cell viability of over 90% even at the maximum treatment dose of 100 µg / mL.

[0054] [Evaluation Example 2] Evaluation of cellular inflammatory response

[0055] Peripheral blood mononuclear cells isolated from normal donor blood were dispersed in a complete culture medium mixed with RPMI 1640, 2 mM L-glutamine, 100 units / ml penicillin, 100 μg / mL streptomycin, and 10% fetal calf serum, and subsequently the cells were plated in 96-cell plates at a 1 x 10 6 Cells were seeded at a cell / well density. Usenamine A was added to cells in a 96-cell culture plate, and the cells were cultured in a CO2 incubator at 37°C for 24 hours. After stimulation with CD3 / CD28, the cells were stained with Pacific Blue-conjugated anti-CD4 (300521, BioLegend) and anti-Fixable Viability Dye-eFluor780 (65-0865-14, Invitrogen). Subsequently, the cells were washed and fixed, infiltrated with Cytofix / Cytoperm buffer, stained with FITC-conjugated anti-IFN-γ (552887, BD) and APC-conjugated anti-IL-17A (512334, BioLegend) antibodies, and analyzed using FlowJo software; the results are shown in Figure 2.

[0056] As shown in Figure 2, as the treatment concentration of Usenamine A increased, the activity of INF-γ, IL-17, and GM-CSF in cells decreased, and it was observed that the reduction effect was particularly high in the 20 μg / mL treatment group. Accordingly, it is believed that Usenamine A inhibits the expression of inflammation-related cytokines.

[0058] [Evaluation Example 3] Evaluation of arthritis in an animal model

[0059] Female SKG mice of the BALB / c lineage were reared in a pathogen-free environment. Five mice were prepared as a positive control (PC), a negative control (NC), and usenamine A treatment groups (0.1, 0.25, and 0.5 mg). Arthritis was induced in mice, excluding the negative control group, by administering 3 mg / kg of Qudlan (Wako, Osaka, Japan) via intraperitoneal injection starting at 8 weeks of age. Prior to arthritis induction, the usenamine A treatment groups were administered 0.1 mg, 0.25 mg, and 0.5 mg of usenamine A, respectively, via a food beverage. Clinical symptoms in the mice were monitored twice a week and scored by two independent observers, as shown in Figure 3.

[0060] According to Figure 3, it can be seen that over time, the arthritis index of the three Usenamine A-treated groups decreased significantly compared to the positive control group (PC) that was not treated with Usenamine A. In addition, since the degree of arthritis relief was greater when the amount of Usenamine A treated was 0.5 mg compared to when it was 0.1 mg or 0.25 mg, it is considered that Usenamine A is effective in treating arthritis.

[0062] As described above, the present invention has been explained by specific details, limited embodiments, and comparative examples; however, these are provided merely to aid in a more comprehensive understanding of the invention, and the invention is not limited to the above embodiments. Those skilled in the art can make various modifications and variations from this description.

[0063] Accordingly, the scope of the present invention is not limited to the described embodiments, and all things equivalent to or having equivalent variations to the claims set forth below, as well as the claims set forth below, shall be considered to fall within the scope of the concept of the present invention.

Claims

Claim 1 A composition for the prevention or treatment of arthritis containing Usenamine A as an active ingredient and inhibiting the secretion of inflammatory cytokines. Claim 2 A composition for the prevention or treatment of arthritis according to claim 1, wherein Usenamine A is extracted from pine needles. Claim 3 A composition for the prevention or treatment of arthritis according to claim 2, wherein the Usenamine A is extracted through a method comprising: a step of extracting dried pine needles using an organic solvent; a step of dissolving the extract in distilled water; a step of fractionating the dissolved extract through chromatography; and a step of obtaining Usenamine A from the fraction. Claim 4 A composition for the prevention or treatment of arthritis according to claim 3, wherein the extraction step comprises performing ultrasonic extraction one or more times. Claim 5 A composition for the prevention or treatment of arthritis according to claim 1, wherein the composition comprises Usenamine A at a concentration of 0.1 to 50 μg / ml. Claim 6 A composition for the prevention or treatment of arthritis according to claim 1, wherein the composition inhibits one or more selected from INF-gamma, IL-17, and GM-CSF. Claim 7 A composition for the prevention or treatment of arthritis according to claim 1, wherein the composition is prepared in any one formulation selected from capsules, topical preparations, powders, granules, tablets, suspensions, emulsions, syrups, and aerosols. Claim 8 A composition for the prevention or treatment of arthritis according to claim 1, wherein the composition further comprises, in addition to the active ingredient, a pharmaceutically acceptable carrier, excipient, or diluent. Claim 9 A health functional food composition for preventing or improving arthritis, containing Usenamine A as an active ingredient and inhibiting the secretion of inflammatory cytokines. Claim 10 A health functional food composition for preventing or improving arthritis according to claim 9, wherein the composition is manufactured in any one formulation selected from powder, granules, pills, tablets, capsules, candies, syrups, and beverages.

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