Composition for prevention or treatment of rheumatoid arthritis comprising supercritical extract of acanthopanax sessiliflorus harms as active ingredient

Abstract

The present invention relates to a composition for the prevention or treatment of rheumatoid arthritis, comprising, as an active ingredient, a supercritical extract of Acanthopanax sessiliflorus Harms. In the present invention, it was confirmed that, upon administration of the supercritical extract of Acanthopanax sessiliflorus Harms to a rheumatoid arthritis animal model, pain induced by rheumatoid arthritis was reduced and cartilage damage was suppressed. Also, a decrease in the infiltration of immune cells in cartilage tissue was confirmed, and it was confirmed that the expression of Col2a1, Sox9, and Aggrecan, which are cartilage anabolic factors whose expression is reduced by inflammatory cytokines and inflammatory factors, was increased, and the expression of Mmp3, Mmp10, Mmp13, and Adamts5, which are cartilage catabolic factors, was reduced. It was also confirmed that pimaric acid and kaurenoic acid, which are useful components of the supercritical extract of Acanthopanax sessiliflorus Harms, protected chondrocytes and suppressed the differentiation of Th17 cells, which are pathogenic cells in rheumatoid arthritis.

Description

Composition for the prevention or treatment of rheumatoid arthritis containing supercritical extract of Acanthopanax japonica as an active ingredient

[0001] The present invention relates to a composition for preventing or treating rheumatoid arthritis, comprising a supercritical extract of Acanthopanax japonica as an active ingredient.

[0002] Rheumatoid arthritis (RA) is a chronic, non-bacterial inflammatory disease characterized by the proliferation of the synovium in joints and tendons. This leads to synovial proliferation and increased synovial fluid, resulting in joint swelling and pain. Rheumatoid arthritis is a systemic inflammatory response that affects many tissues and organs (skin, blood vessels, heart, lungs, muscles) throughout the body, but particularly affects joints, causing irreversible proliferative synovitis that progresses to the destruction of articular cartilage and joint stiffness. While the cause of RA remains unknown, an autoimmune response is known to play a key role in the chronicity and progression of the disease. This persistent autoimmune response, characterized by the local release of inflammatory mediators and cytokines, along with the crucial role of T cells, ultimately destroys joints. Key symptoms include fatigue, weakness, and pain. As arthritis progresses, fever and physical weakness may also occur. Additionally, muscle atrophy and muscle spasm may occur around the inflamed joint, affecting joint movement.

[0003] Therefore, the goal of treating arthritis is not only to reduce joint pain and inflammation and prevent joint deformation, but more fundamentally, to elucidate and control the intracellular mechanisms that trigger arthritis. However, despite extensive research into the causes and mechanisms of rheumatoid arthritis and the development of various new drugs utilizing these findings, a treatment that achieves a complete cure has yet to be developed.

[0004] Currently known treatments for arthritis include nonsteroidal anti-inflammatory drugs (NSAIDs) (aspirin and ibuprofen), gold, penicillamine, and steroid hormones. However, these treatments are associated with side effects when taken long-term. Recently, efforts have been made to develop a therapeutic using genetic recombination technology to produce a soluble receptor for tumor necrosis factor (TNF), a key player in the inflammatory process. However, these methods have resulted in unexpected side effects, including inflammation, edema, abnormal neovascularization, and erosion of bone and cartilage tissue.

[0005] Meanwhile, Acanthopanax sessiliflorusHarms is a deciduous shrub in the Araliaceae family, belonging to the Acanthopanax genus. About 35 species of Acanthopanax plants have been reported worldwide, and seven species are known to grow wild in Korea, including Acanthopanax tree, Island Acanthopanax, Seoul Acanthopanax, Jirisan Acanthopanax, Hair Acanthopanax, Gasis Acanthopanax, and King Acanthopanax. Its roots, rhizomes, and bark have been used as medicinal herbs in folk and oriental medicine since ancient times, and contain various glycosides such as carotene, ligustrin, and galactoside, as well as flavone, chlorogenic acid, sesamin, caffeic acid, essential oils, and polysaccharides. The pharmacological effects of Eleutherococcus japonica include sedative, antioxidant, anti-stress, immune-boosting, and muscle-relaxing effects. It is also used as a treatment for conditions such as lumbago, numbness in the hands and feet, arthritis, stroke, high blood pressure, and cancer. Furthermore, due to its excellent efficacy and lack of reported side effects such as insomnia, efforts are continuously being made to utilize it as a pharmaceutical or functional food.

[0006] Meanwhile, "supercritical extraction" (also known as supercritical fluid extraction) is an extraction process using supercritical fluids, such as carbon dioxide and pentane. Among these, supercritical extraction (Dictionary of Chemical Terms, January 15, 2011, Iljinsa) is particularly notable for its low critical point (31.1℃, 73.8atm) and non-toxicity, making it promising for the effective extraction and separation of useful substances. It is widely used in the food industry for the extraction of hops and caffeine. It utilizes the properties of liquids, gases, and fluids to facilitate extraction and separation processes. Its low surface tension allows for easy penetration into pore structures, excellent diffusion, and strong solubility, making it an efficient extraction process. Furthermore, it is an environmentally and human-friendly, clean technology applicable to high-purity, high-quality product processes. Its supercritical properties enable the stable production of selective extraction and thermally denatured materials at low temperatures. The advantages of supercritical pine nut extraction are that the heating temperature is lower than that of conventional extraction methods, so there is less risk of color and component denaturation, the rate of oxidation can be reduced, and the amount of effective ingredients extracted is large.

[0007] Accordingly, the inventors of the present invention confirmed that the supercritical extract of Acanthopanax spp. improves rheumatoid arthritis symptoms and protects joints in an animal model of rheumatoid arthritis, thereby completing the present invention.

[0008] The purpose of the present invention is to provide a pharmaceutical composition for preventing or treating rheumatoid arthritis, which comprises a supercritical extract of Acanthopanax sessiliflorusHarms as an active ingredient.

[0009] Another object of the present invention is to provide a food composition for preventing or improving rheumatoid arthritis, which contains a supercritical extract of Acanthopanax sessiliflorusHarms as an active ingredient.

[0010] Another object of the present invention is to provide a method for treating rheumatoid arthritis, comprising the step of administering to a subject a pharmaceutically acceptable amount of a supercritical extract of Acanthopanax sessiliflorusHarms.

[0011] In order to achieve the above purpose, the present invention provides a pharmaceutical composition for preventing or treating rheumatoid arthritis, which comprises a supercritical extract of Acanthopanax sessiliflorusHarms as an active ingredient.

[0012] In addition, the present invention provides a food composition for preventing or improving rheumatoid arthritis, which contains a supercritical extract of Acanthopanax sessiliflorusHarms as an active ingredient.

[0013] In addition, the present invention provides a method for treating rheumatoid arthritis, comprising the step of administering to a subject a pharmaceutically acceptable amount of a supercritical extract of Acanthopanax sessiliflorusHarms.

[0014] The present invention confirmed that when a supercritical extract of Eleutherococcus japonica is administered to an animal model of rheumatoid arthritis, pain induced by rheumatoid arthritis is reduced and cartilage damage is inhibited. In addition, it was confirmed that infiltration of immune cells into cartilage tissue is reduced, and the expression of Col2a1, Sox9, and Aggrecan, which are cartilage anabolic factors whose expression is reduced by inflammatory cytokines and inflammatory factors, is increased, and the expression of Mmp3, Mmp10, Mmp13, and Adamts5, which are cartilage catabolic factors, is decreased. In addition, it was confirmed that pimaric acid and kaurenoic acid, which are useful components of the supercritical extract of Eleutherococcus japonica, protect cartilage cells and inhibit the differentiation of Th17 cells, which are pathogenic cells of rheumatoid arthritis, and therefore, the present invention can be usefully utilized in related industries.

[0015] Figure 1 is a diagram showing the extraction method and analysis of useful components of the supercritical extract of the present invention using CG-MS (A: schematic diagram of the extraction process, B: GC-MS results).

[0016] Figure 2 is a diagram showing the clinical symptoms of arthritis confirmed by administering the supercritical extract of the present invention to an animal model of rheumatoid arthritis.

[0017] A: Schematic diagram of the experimental process

[0018] B: Visual observation of hind legs and lymph nodes

[0019] C: Quantification of clinical indices

[0020] D: Quantification of plantar edema

[0021] Figure 3 is a diagram showing the analysis of motility according to the induction of arthritis by administering the supercritical extract of the present invention to an animal model of rheumatoid arthritis using a hot plate method (A: confirmation of motility by week, B: quantification of motility by week).

[0022] Figure 4 is a diagram showing histological analysis of the degree of joint damage by administering the supercritical extract of the present invention to an animal model of rheumatoid arthritis (A: knee tissue staining results, B: knee damage quantification, C: ankle tissue staining results, D: ankle damage quantification, E: toe tissue staining results, F: toe damage quantification).

[0023] Figure 5 is a diagram showing histological analysis of pannus formation in the knee and toes by administering the supercritical extract of the present invention to an animal model of rheumatoid arthritis (A: knee tissue staining results, B: quantification of knee tissue pannus formation, C: toe tissue staining results, D: quantification of toe tissue pannus formation).

[0024] Figure 6 is a diagram showing the infiltration of immune cells into cartilage tissue by administering the supercritical extract of the present invention to an animal model of rheumatoid arthritis.

[0025] A: Eosinophil staining results

[0026] B: Quantification of eosinophil infiltration

[0027] C: Neutrophil staining results

[0028] D: Quantification of neutrophil infiltration

[0029] E: Mast cell staining results

[0030] F: Quantification of mast cell infiltration

[0031] Figure 7 is a diagram showing the expression of cartilage metabolic factors analyzed by RT-PCR after treating chondrocytes treated with an inflammatory factor with the supercritical extract of the present invention.

[0032] Figure 8 is a diagram showing the results of Western blot analysis of the inhibition of NO production and the inhibition of the expression of inflammatory proteins by treating LPS-treated cartilage cells with the supercritical extract of the present invention.

[0033] Figure 9 is a diagram showing the expression of cartilage metabolic factors analyzed by RT-PCR by treating cartilage cells treated with an inflammatory factor with the useful components of the supercritical extract of the present invention.

[0034] Figure 10 is a diagram showing the effect of controlling differentiation of TH17 cells by treating splenocytes induced to differentiate into TH17 cells with the useful components of the supercritical extract of the present invention using flow cytometry.

[0035] Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. In the following description, detailed descriptions of well-known technologies to those skilled in the art may be omitted. Furthermore, in describing the present invention, detailed descriptions of related known functions or configurations may be omitted if it is determined that such detailed descriptions may unnecessarily obscure the gist of the present invention. Furthermore, the terminology used in this specification is intended to appropriately express preferred embodiments of the present invention, and may vary depending on the intentions of the user or operator, or the customs of the field to which the present invention pertains.

[0036] Therefore, definitions of these terms should be based on the overall content of this specification. Throughout this specification, whenever a part is said to "include" a component, this does not exclude other components, but rather implies the inclusion of additional components, unless otherwise specifically stated.

[0037] The present invention provides a pharmaceutical composition for preventing or treating rheumatoid arthritis, comprising a supercritical extract of Acanthopanax sessiliflorusHarms as an active ingredient.

[0038] The term “prevention” as used in the present invention means any act of suppressing symptoms or delaying progression of a specific disease by administering the composition of the present invention.

[0039] The term "treatment" as used in the present invention means any act of improving or beneficially altering the symptoms of a specific disease by administering the composition of the present invention.

[0040] The pharmaceutical composition of the present invention may further include an adjuvant in addition to the active ingredient. Any adjuvant known in the art may be used without limitation. However, for example, Freund's complete adjuvant or incomplete adjuvant may be further included to enhance its effectiveness.

[0041] The pharmaceutical composition according to the present invention can be prepared in a form in which the active ingredient is mixed with a pharmaceutically acceptable carrier. Here, the pharmaceutically acceptable carrier includes carriers, excipients, and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0042] The pharmaceutical composition of the present invention can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injection solutions, each according to a conventional method.

[0043] When formulated, it can be prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants that are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and such solid preparations can be prepared by mixing the active ingredient with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups, and in addition to commonly used diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives can be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, Tween 61, cocoa butter, laurin, and glycerogelatin.

[0044] The pharmaceutical composition according to the present invention can be administered to a subject via various routes. All modes of administration are contemplated, including oral, intravenous, intramuscular, subcutaneous, and intraperitoneal injection.

[0045] The dosage of the pharmaceutical composition according to the present invention is selected in consideration of the age, weight, sex, physical condition, etc. of the subject. It is obvious that the concentration of the active ingredient included in the pharmaceutical composition can be selected in various ways depending on the subject, and it is preferably included in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg / ml. If the concentration is less than 0.01 μg / ml, pharmaceutical activity may not be observed, and if it exceeds 5,000 μg / ml, it may be toxic to the human body.

[0046] According to one embodiment of the present invention, the supercritical extract may be extracted by carbon dioxide (CO2) supercritical extraction.

[0047] According to one embodiment of the present invention, the extract may include a compound of the following chemical formula 1.

[0048] [Chemical Formula 1]

[0049]

[0050] The compound of the above chemical formula 1 of the present invention is named pimaric acid and is a compound with CAS number 127-27-5.

[0051] According to one embodiment of the present invention, the extract may include a compound of the following chemical formula 2.

[0052] [Chemical Formula 2]

[0053]

[0054] The compound of the above chemical formula 2 of the present invention is named kaurenoic acid and is a compound with CAS number 6730-83-2.

[0055] According to one embodiment of the present invention, the extract may reduce pain induced by rheumatoid arthritis.

[0056] According to one embodiment of the present invention, the extract may inhibit cartilage damage, and the cartilage damage may be synovitis, pannus formation, or cartilage degeneration.

[0057] According to one embodiment of the present invention, the cartilage damage may be an increase in infiltration of eosinophils, neutrophils or mast cells into cartilage cells.

[0058] According to one embodiment of the present invention, the extract may regulate the expression of a cartilage metabolic factor, and the regulation of the expression of the cartilage metabolic factor may be to suppress the expression of a cartilage catabolic factor selected from the group consisting of matrix metalloproteinase-3 (Mmp3), Mmp10, Mmp13, and Adamts5 (A disintegrin and metalloproteinase with thrombospondin motifs 5), and the cartilage catabolic factor may be one whose expression is induced by an inflammatory factor.

[0059] According to one embodiment of the present invention, the regulation of expression of the cartilage metabolic factor may be to increase the expression of collagen type II alpha 1 (Col2a1), a cartilage anabolic factor, transcription factor SOX-9 (Sox9), or aggrecan, and the cartilage anabolic factor may be an inflammatory factor whose expression is reduced.

[0060] According to one embodiment of the present invention, the inflammatory factor may be IL-1β, TNF-α or LPS.

[0061] According to one embodiment of the present invention, the extract may inhibit differentiation of Th17 cells.

[0062] In addition, the present invention provides a food composition for preventing or improving rheumatoid arthritis, which contains a supercritical extract of Acanthopanax sessiliflorusHarms as an active ingredient.

[0063] The term "improvement" as used herein means any action that at least reduces a parameter associated with the condition being treated, for example, the severity of a symptom.

[0064] The food composition of the present invention may contain, in addition to containing the effective ingredient of the present invention, various flavoring agents or natural carbohydrates as additional ingredients, like conventional food compositions.

[0065] Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, etc.; and polysaccharides such as dextrin, cyclodextrin, etc., and common sugars, and sugar alcohols such as xylitol, sorbitol, erythritol, etc. As the above-mentioned flavoring agent, natural flavoring agent (thaumatin), stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agent (saccharin, aspartame, etc.) can be advantageously used. The food composition of the present invention can be formulated in the same manner as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes, and health supplements.

[0066] In addition, the food composition may contain, in addition to the extract as an active ingredient, various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the food composition of the present invention may contain fruit pulp for producing natural fruit juice, fruit juice drinks, and vegetable drinks.

[0067] The functional food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of preventing or treating rheumatoid arthritis. The term "health functional food composition" in the present invention refers to a food manufactured and processed using raw materials or ingredients having functionality useful to the human body according to Act No. 6727 on Health Functional Foods, and means to be consumed for the purpose of obtaining a useful effect for health purposes such as regulating nutrients for the structure and function of the human body or physiological effects. The health functional food of the present invention may include conventional food additives, and whether it is suitable as a food additive is determined by the specifications and standards for the relevant item according to the general provisions and general test methods of the Food Additives Codex approved by the Ministry of Food and Drug Safety, unless otherwise specified. Items listed in the "Food Additives Codex" include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Examples thereof include natural additives such as persimmon pigment, licorice extract, crystalline cellulose, high-molecular weight pigment, and guar gum; mixed preparations such as sodium L-glutamate preparations, noodle additive alkaline agents, preservative preparations, and tar color preparations. For example, a health functional food in tablet form can be prepared by mixing the active ingredient of the present invention with excipients, binders, disintegrants, and other additives, granulating the mixture using a conventional method, and then adding a lubricant, etc. to compress and molding, or directly compressing and molding the mixture. In addition, the health functional food in tablet form can contain a maturing agent, etc., if necessary. Among health functional foods in capsule form, hard capsules can be prepared by filling a mixture of the active ingredient of the present invention with additives such as excipients into a conventional hard capsule, and soft capsules can be prepared by filling a mixture of the active ingredient of the present invention with additives such as excipients into a capsule base such as gelatin. The above soft capsule may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative, etc., as needed.The ring-shaped health functional food can be prepared by molding a mixture of the active ingredient of the present invention with excipients, binders, disintegrants, etc. using a conventionally known method, and, if necessary, can be coated with white sugar or other coating agents, or the surface can be coated with a substance such as starch or talc. The granular health functional food can be manufactured into a granular form using a mixture of the active ingredient of the present invention with excipients, binders, disintegrants, etc. using a conventionally known method, and, if necessary, can contain a flavoring agent, a flavoring agent, etc.

[0068] In addition, the present invention provides a method for treating rheumatoid arthritis, comprising the step of administering to a subject a pharmaceutically acceptable amount of a supercritical extract of Acanthopanax sessiliflorusHarms.

[0069] The treatment method of the present invention comprises administering to a subject a therapeutically effective amount of the supercritical extract of Acanthopanax senticosus. The specific therapeutically effective amount for a specific subject is preferably applied differently depending on various factors including the type and degree of response to be achieved, the specific composition including whether other agents are used in some cases, the age, body weight, general health, sex and diet of the subject, the time of administration, the route of administration and the secretion rate of the composition, the treatment period, drugs used together or simultaneously with the specific composition, and similar factors well known in the medical field. The daily dosage is 0.0001 to 100 mg / kg, preferably 0.01 to 100 mg / kg, based on the amount of the pharmaceutical composition of the present invention, and can be administered 1 to 6 times a day. However, it is obvious to those skilled in the art that the dosage or administration of each active ingredient should be such that the content of each active ingredient is not excessively high and side effects are not caused. Therefore, the effective amount of a composition suitable for the purpose of the present invention is preferably determined in consideration of the aforementioned matters.

[0070] The above object is applicable to any mammal, which includes not only humans and primates, but also livestock such as cows, pigs, sheep, horses, dogs and cats.

[0071] The supercritical extract of the present invention can be administered to mammals such as rats, mice, livestock, and humans via various routes. All modes of administration are conceivable, and for example, it can be administered orally, rectally, or by intravenous, intramuscular, subcutaneous, intrauterine, or intracerebroventricular injection.

[0072] Hereinafter, the present invention will be described in more detail with reference to examples. These examples are intended merely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

[0073] <Example 1> Preparation for confirming the antirheumatic effect of supercritical extract of Acanthopanax japonica

[0074] <1-1> Preparation of supercritical extract of Acanthopanax japonica

[0075] In order to confirm the effect of the supercritical extract of Acanthopanax sessiliflorus Harms on improving rheumatoid arthritis of the present invention, the supercritical extract of Acanthopanax sessiliflorus was prepared. Specifically, carbon dioxide supercritical extraction was used, and the extraction device (Nantong Wisdom Supercritical Science and Technology Development Co., Ltd, China) consisting of a 5 L extractor, two separators (separator 1:3 L, separator 2:2 L), a condenser, and a high-pressure CO2 pump that supplies CO2 to the system from a CO2 cylinder was used. All devices were heated using a heating jacket, and all containers were heated. Then, 2 kg of powdered Acanthopanax sessiliflorus Harms was loaded into the extractor, and CO2 was pumped. Thereafter, valve 9 (V9) was opened to empty the device, and three CO2 purges were performed to replenish the air in the device. Afterwards, all valves except V1 and V2 were closed after purging so that a certain amount of CO2 could be injected into the extractor. After the pressure and temperature of the extractor reached 40 MPa and 55°C, respectively, V5, V8, and V11 were opened for CO2 circulation and dynamic extraction. Separators 1 and 2 were then adjusted to 50°C, 13 MPa, and 40°C, 5 MPa, respectively. The CO2 mass flow was maintained at 35 kg / h during the 2-hour extraction process. Afterwards, V7 was used to collect samples and prepare the supercritical extract (ASH) of Acanthopanax spp.

[0076] <1-2> Analysis of useful components of supercritical extract of Acanthopanax japonica

[0077] In order to analyze the useful components of the supercritical extract of Acanthopanax spp. of the present invention, gas chromatography-mass spectrometry (GC-MS) was used. Specifically, the supercritical extract of Acanthopanax spp. of Example 1-1 was analyzed using a Shimadzu QP2010 SE gas chromatography-mass spectrometer (Shimadzu, Kyoto, Japan) equipped with a DB-1 capillary column (length 25 m × diameter 0.32 mm × thickness 0.52 μm), and helium was used as a carrier gas at a constant flow rate of 0.5 mL / min. The GC-MS spectrum was detected by the electron ionization energy method with a high ionization energy of 70 eV (electron volts), and the injector temperature was set to 250°C. The column temperature was set at 40°C for 4 minutes and then increased at 10°C per minute to 240°C. The retention time (minutes), peak area, peak height, and mass spectrum of phytochemicals contained in the supercritical extract of E. schizontii were compared with the mass spectral library database to quantify the phytochemical content in the supercritical extract of E. schizontii.

[0078] <1-3> Animal model of rheumatoid arthritis

[0079] In order to confirm the effect of the supercritical extract of the present invention on improving rheumatoid arthritis (RA), an animal model of rheumatoid arthritis was created, and its disease activity was evaluated. Specifically, 7-week-old DBA / 1J male mice were used to create an RA model, and the mice were anesthetized with isoprene, and a mixture of 4 mg / ml of collagen type II and an equal amount of complete Freund's adjuvant was injected into the tail skin to create a collagenase-induced arthritis (CIA) animal model. Thereafter, von Frey and hot plate assays were performed to evaluate disease activity, and the von Frey assay was performed once a week for 7 days after arthritis induction. Specifically, the pain of the mice was confirmed using the von Frey assay, and the threshold of the response was confirmed starting from the size of the thickest filament. Additionally, the motility of the mice was determined by hot plate analysis. Using a Stimulation-Hargreaves Apparatus (ugo basile, 37570, Gemonio, Italy), the mice were placed at five different points on the plate, and the time taken for the mice to move on the plate was recorded in seconds.

[0080] In addition, the supercritical extract of the present invention was injected intraperitoneally one week after inducing rheumatoid arthritis, and ASH was dissolved in polyethylene glycol 400 (PEG-400) at a concentration of 200 μg / ml and injected intraperitoneally twice a week. As a control group, 200 μl of PEG-400 was injected intraperitoneally.

[0081] <1-4> Check clinical indices and foot thickness

[0082] One week after CIA induction, the degree of hind paw swelling was assessed at 3-4-day intervals. Both paws were measured relative to the mouse's hind paw, and clinical scores were calculated using the Hooke Laboratories' severity index. The clinical score scale is as follows:

[0083] 0 points: normal

[0084] 1 point: Swollen 1-2 toes or no foot swelling

[0085] 2 points: Swollen three or more toes or slight swelling of the entire foot

[0086] 3 points: Increased swelling of the genitals

[0087] 4 points: Severe swelling of the developing body and inability to hold the toes.

[0088] <1-5> Histological analysis

[0089] For histological analysis, tissues obtained from each group were fixed in 4% paraformaldehyde for 24 hours before being decalcified in 0.5 M ethylenediaminetetraacetic acid (pH 7.4) for 2 weeks. The tissues were then sectioned into 4 μm thick sections, embedded in paraffin, and paraffin blocks were prepared for slide preparation. Histological examinations were stained using Harris hematoxylin, fast green, and safranin O (all Sigma-Aldrich, St. Louis, MO, USA). The degree of histological inflammation in RA was indexed into three categories: OARSI grade (0–6), synovitis score (0–3), and pannus score (0–3).

[0090] In addition, to analyze the infiltration of immune cells, the infiltration of eosinophils and mast cells into the synovial membrane in the joint space was confirmed. Eosinophils were stained with Congo red (Sigma C6277, St. Louis, MO, USA), and mast cells were stained with toluidine blue O (Sigma-Aldrich). Afterwards, neutrophils were quantified using ImageJ software (NIH), and CD15 was applied to the neutrophil extracellular trap.

[0091] <1-6> RT PCR analysis

[0092] For reverse transcription-polymerase chain reaction (RT-PCR) analysis, total RNA was extracted from primary cultured chondrocytes (Molecular Research Center Inc., Cincinnati, OH, USA) using TRIzol reagent. The purity and concentration of RNA were confirmed using a NanoDropTM 2000 Spectrophotometer (Thermo Scientific, Waltham, MA, USA). RNA was then synthesized into cDNA and amplified using a CFX96TM Real-Time System (Bio-Rad Laboratories, Inc., Hercules, CA, USA, at the Bio-Health Materials Core-Facility, Jeju National University, CA) and SYBR Premix ExTaq reagent (TaKaRa Bio, Mountain View, CA, USA). GAPDH was used as an internal control, and the primers used for PCR amplification are shown in Table 1 below.

[0093] [Table 1]

[0094]

[0095] <1-7> Western blot analysis

[0096] The protein content of each sample was quantified using BCA analysis, and 50 μg of protein per sample was separated using 10-12% Bis-Tris gels (Bio-Rad Laboratories, Inc.). The separated proteins were then transferred to Odyssey Nitrocellulose Membrane (LI-COR, Lincoln, NE, USA), blocked with Intercept (TBS) Blocking Buffer (LI-COR), and incubated with each primary antibody at 4°C for 12-16 hours. Secondary antibodies were then added and incubated in the dark at 20°C for 1 hour. The expressed protein bands were then identified using the Odyssey DLx Imaging System (LI-COR).

[0097] <1-9> Statistical analysis

[0098] All statistical analyses were performed using IBM SPSS Statistics software (IBM Corp., Armonk, NY, USA). To compare data from cell-based in vitro experiments for pairwise and multiple comparisons, two-tailed Student's t-tests and two-way analysis of variance with a post hoc test (LSD) were used, respectively, considering sample sizes and unequal variances. OARSI grade, bone tissue maturity, and synovitis data obtained from histological experiments were analyzed using the nonparametric Mann-Whitney U test, and the Shapiro-Wilk test was used to check for normality of distribution. The cutoff for statistical significance was set at P <0.05.

[0099] <Example 2> Confirmation of useful components of supercritical extract of Achyranthes japonica

[0100] As a result of GC-MS analysis of the supercritical extract of E. gallica, 10 peaks containing physiologically active compounds were identified (Table 2). Various compounds identified by peak retention time, peak area (%), height (%), and mass spectrum were compared with a spectral library. As a result of peak area and library comparison, pimaric acid and kaurenoic acid were confirmed as the two major compounds among the 10 compounds as the major compounds of the supercritical extract of E. gallica. The specific extraction method and compound identification process are shown in Figure 1.

[0101] [Table 2]

[0102]

[0103] <Example 3> Confirmation of the pain-suppressing effect of the supercritical extract of Acanthopanax japonica on rheumatoid arthritis

[0104] The ASH group treated with the supercritical extract of Acanthopanax spp. showed a lower severity of rheumatoid arthritis compared to the control group treated with PEG alone. The specific experimental process is shown in Figure 2A, and the clinical RA index was significantly lower in the ASH group than in the PEG alone group (Figure 2B). In addition, the clinical score and paw edema induced by CIA were significantly lower in the ASH group (Figures 2C and 2D). Furthermore, a hot plate analysis after immunization with collagen confirmed that the latency period was reduced in the ASH group compared to the CIA group (Figure 3), and the analgesic effect on pain induced by rheumatoid arthritis was statistically significant at weeks 2, 3, and 4 after ASH administration.

[0105] <Example 4> Confirmation of cartilage protection by supercritical extract of Acanthopanax japonica

[0106] In the ASH-treated group, we confirmed that CIA-induced synovitis, pannus formation, and cartilage degeneration were reduced. Specifically, the degree of cartilage damage, synovial inflammation, and infiltration of inflammatory mediator cells were confirmed using safranin O staining (Figures 4A, 4C, and 4E). According to the OARSI grade, ASH administration significantly reduced cartilage damage in the knee, ankle, and toes (Figures 4B, 4D, and 4F). In addition, safranin O staining of knee tissues showed that pannus formation was significantly reduced in ASH-treated mice compared to PEG-treated mice (Figure 5).

[0107] <Example 5> Confirmation of inhibition of immune cell infiltration by supercritical extract of Acanthopanax japonica

[0108] Eosinophil infiltration in the synovial tissue of the CIA group was confirmed by Congo red staining (Fig. 6A). The number of eosinophils was significantly decreased in the ASH group compared to the PEG group (Fig. 6B). CDr15 immunostaining confirmed that neutrophil infiltration increased in the PEG group, and the infiltrated neutrophils were decreased in the ASH-treated group (Fig. 6D). In addition, mast cell infiltration was confirmed by toluidine blue staining, and mast cells were stained (Fig. 6E). The number of mast cells was decreased in the ASH group compared to the CIA group, and degranulated mast cells were significantly decreased in the ASG group compared to the CIA group (Fig. 6F).

[0109] <Example 6> Confirmation of the inflammatory cytokine regulation effect of the supercritical extract of Acanthopanax japonica

[0110] <6-1> Confirmation of cytokine-induced cartilage anabolism and catabolic factor regulation

[0111] We confirmed the expression of cartilage anabolic and catabolic proteins induced by proinflammatory cytokines and inflammatory mediators of the supercritical extract of E. gallica. Specifically, we confirmed that proinflammatory cytokines IL-1β, TNF-α, and LPS decreased the expression of protein anabolic factors Col2a1, SOX9, and Aggrecan, and increased the expression of protein catabolic factors Mmp3, Mmp13, Mmp10, and Adamts5 in primary cultured mouse chondrocytes (Figures 7A-7C). However, when ASH was treated with IL-1β, the decreased expression of Col2a1, Sox9, and Aggrecan was increased (Figure 7D), and TNF-α-mediated Adamts5 expression was reduced (Figure 7E). In addition, ASH was confirmed to decrease LPS-mediated Mmp3, Mmp10, Mmp13, and Adamts5 expression, and to restore LPS-mediated inhibition of SOX9 expression (Figure 7F).

[0112] <6-2> Confirmation of inhibition of NO production and NF-κB activation

[0113] We examined whether ASH treatment could suppress NO production in relation to NF-κB activity. Compared to the LPS-only treatment group, NO production was significantly reduced in proportion to the concentration of the ASH extract (Fig. 8A). After ASH treatment, the expression of factors related to NF-κB activity showed that the production of inflammatory enzymes iNOS and COX-2 significantly decreased as the concentration of ASH increased (Fig. 8B). In addition, while LPS treatment increased the phosphorylation of IκBa, ASH treatment inhibited the phosphorylation of IκBa and the phosphorylation of NF-κB p65 (Figs. 8C and 8D). These results confirmed that the supercritical extract of Eleutherococcus spp. suppresses NO production and inflammatory responses by inhibiting the activation of NF-κB in an inflammatory environment.

[0114] <6-3> Confirmation of the cartilage catabolism inhibitory effects of pimaric acid and kaurenoic acid

[0115] The anti-inflammatory effects of pimaric acid and kaurenoic acid, which are useful components of the supercritical extract of E. gallicum, were confirmed in chondrocytes induced with inflammation by pro-inflammatory cytokines IL-1β, TNF-α, and LPS. The expression of cartilage anabolic factors Col2a1, Sox9, and Aggrecan increased with pimaric acid treatment, while the expression of cartilage catabolic factors MMP-3, MMP-13, Adamts4, and Adamts5 decreased (Figs. 9A to 9C). In addition, kaurenoic acid was confirmed to regulate the expression of the above factors (Figs. 9D to F), confirming that pimaric acid and kaurenoic acid, which are useful components of the supercritical extract of E. gallicum, have cartilage protective effects.

[0116] <Example 7> Confirmation of the T cell regulatory effects of pimaric acid and kaurenoic acid.

[0117] Since IL-17A is clinically associated with rheumatoid arthritis patients, we investigated whether pimaric acid and kaurenoic acid affect the differentiation of CD4-positive T cells (Th17), which are IL-17A-producing T cells. Specifically, splenocytes from C57BL / 6 mice were stimulated with anti-CD3 / CD28 and Th17-polarizing cytokines IL-6 and TGF-β, and a significant increase in the expression of IL-17A-positive cells among CD4-positive T cells was confirmed in the stimulated cells (Fig. 10). However, treatment with pimaric acid at a concentration of 50 μg / ml confirmed a 2.5-fold decrease in the number of IL-17A-positive cells. In addition, similar to pimaric acid, treatment with kaurenoic acid also confirmed that the expression of IL-17A positive cells in CD4 positive T cells was significantly reduced, confirming that pimaric acid and kaurenoic acid, which are useful components in the supercritical extract of Acanthopanax galliculata, regulate the differentiation of TH17 cells, which are pathogenic cells of rheumatoid arthritis.

[0118] Accordingly, the present invention confirmed that when a supercritical extract of Echinococcus japonica was administered to an animal model of rheumatoid arthritis, pain induced by rheumatoid arthritis was reduced and cartilage damage was inhibited. In addition, it was confirmed that infiltration of immune cells into cartilage tissue was reduced, and the expression of Col2a1, Sox9, and Aggrecan, which are cartilage anabolic factors whose expression is reduced by inflammatory cytokines and inflammatory factors, was increased, and the expression of Mmp3, Mmp10, Mmp13, and Adamts5, which are cartilage catabolic factors, was decreased. In addition, it was confirmed that pimaric acid and kaurenoic acid, which are useful components of the supercritical extract of Echinococcus japonica, protect cartilage cells and inhibit the differentiation of Th17 cells, which are pathogenic cells of rheumatoid arthritis.

Claims

1. A pharmaceutical composition for the prevention or treatment of rheumatoid arthritis, comprising a supercritical extract of Acanthopanax sessiliflorusHarms as an active ingredient.

2. In paragraph 1, A composition wherein the supercritical extract is extracted by carbon dioxide (CO2) supercritical extraction.

3. In paragraph 1, The above extract is a composition comprising a compound of the following chemical formula 1. [Chemical Formula 1] .

4. In paragraph 1, The above extract is a composition comprising a compound of the following chemical formula 2. [Chemical Formula 2] .

5. In paragraph 1, A composition wherein the extract reduces pain induced by rheumatoid arthritis.

6. In paragraph 1, A composition wherein the above extract inhibits cartilage damage.

7. In paragraph 6, A composition wherein the above cartilage damage is synovitis, pannus formation or cartilage degeneration.

8. In paragraph 6, The above cartilage damage is a composition in which there is increased infiltration of eosinophils, neutrophils or mast cells into cartilage cells.

9. In paragraph 1, A composition wherein the above extract regulates the expression of cartilage metabolic factors.

10. In paragraph 9, A composition for regulating the expression of the above cartilage metabolic factor, wherein the composition suppresses the expression of a cartilage catabolic factor selected from the group consisting of matrix metalloproteinase-3 (Mmp3), Mmp10, Mmp13, and Adamts5 (A disintegrin and metalloproteinase with thrombospondin motifs 5).

11. In paragraph 10, A composition wherein the above cartilage catabolic factor is induced to be expressed by an inflammatory factor.

12. In paragraph 11, A composition wherein the inflammatory factor is IL-1β, TNF-α or LPS.

13. In paragraph 9, A composition in which the expression regulation of the above cartilage metabolic factor increases the expression of collagen type II alpha 1 (Col2a1), a cartilage anabolic factor, transcription factor SOX-9 (Sox9), or aggrecan.

14. In paragraph 13, A composition wherein the above cartilage assimilation factor is a composition in which the expression of the inflammatory factor is reduced.

15. In paragraph 1, The above extract is a composition that inhibits differentiation of Th17 cells.

16. A food composition for preventing or improving rheumatoid arthritis, containing a supercritical extract of Acanthopanax sessiliflorusHarms as an active ingredient.

17. A method for treating rheumatoid arthritis, comprising administering to a subject a pharmaceutically acceptable amount of a supercritical extract of Acanthopanax sessiliflorusHarms.

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