Composition for treating osteoarthritis containing herbal medicine extract as active ingredient

Abstract

The present invention relates to a composition for treating osteoarthritis, the composition containing an herbal medicine extract as an active ingredient. It was found that an herbal medicine complex extract according to the present invention improves an arthritis index and increases bone mineral mass and bone density in an osteoarthritis animal model. In addition, it was found that the herbal medicine complex extract prevents cartilage damage, thereby increasing the thickness of cartilage, and regulates the expression of tissue regeneration factors and inflammatory cytokines.

Description

Composition for treating osteoarthritis containing herbal extracts as active ingredients

[0001] The present invention relates to a composition for treating osteoarthritis comprising a herbal extract as an active ingredient.

[0002]

[0003] Arthritis is broadly categorized into osteoarthritis and rheumatoid arthritis. Osteoarthritis, also known as degenerative arthritis, is the most common form of arthritis. It primarily occurs in middle-aged or older adults and causes pain, stiffness, and limited movement in weight-bearing joints, such as the knees and hips. While previously thought to be simply a consequence of aging, it is now believed that the symptoms vary due to a complex mix of factors, including age, genetics, obesity, joint shape, and hormones.

[0004] It is known that degenerative arthritis is mainly caused by chronic inflammation due to gradual damage to articular cartilage, which in turn leads to degeneration of surrounding tissues. More specifically, when transcription factors such as NF-kB and AP-1 are activated in cartilage tissue due to accumulated physical stress, chronic inflammation occurs, and factors that mediate and amplify inflammatory responses such as COX-2 and LOX are expressed, surrounding blood flow is suppressed, and tissue-destructive factors such as matrix metalloproteinases (MMPs), hyaluronidase, and iNOS are overexpressed, destroying cartilage tissue. As a result, surrounding tissues such as muscles, tendons, and ligaments are affected, causing severe pain.

[0005] Rheumatoid arthritis is an inflammatory autoimmune disease that affects multiple joints. The synovial tissue and fluid of patients with rheumatoid arthritis are characterized by excessive activity of inflammatory cells (macrophages, T cells, B cells, dendritic cells, etc.), causing chronic inflammation, joint and cartilage damage, and pain.

[0006] To date, there is no treatment for arthritis, and non-steroidal anti-inflammatory drugs (NSAIDs) are generally used to relieve joint inflammation. However, since NSAIDs only temporarily relieve joint inflammation, they are not an appropriate treatment for degenerative arthritis, a non-inflammatory arthritis that requires promotion of cartilage formation and inhibition of cartilage tissue destruction (Pritchard MH et al., Annals of the Rheumatic Diseases, 37:493-503, 1978). These non-steroidal anti-inflammatory drugs (NSAIDs) are suitable for blocking the inflammatory mechanism as a treatment for rheumatoid arthritis, an inflammatory arthritis, but they have been pointed out as problems in accelerating cartilage damage and causing side effects in the cardiovascular, gastrointestinal, kidney, and liver.

[0007] In addition, the autologous chondrocyte transplantation technique currently developed for cartilage formation involves harvesting the cartilage and subchondral bone already formed in the patient's normal area, digging an appropriate hole over the damaged cartilage area, and transplanting it to create hyaline cartilage. This has been successful in some patients, but it cannot be a universal method because it can only be performed on patients with a small area of ​​cartilage tissue destruction and who are eligible for autologous transplantation (Peterson L et al., J Bone Joint Surg Am. 85-A Suppl:17-24, 2003).

[0008] Accordingly, the inventors of the present invention confirmed that a herbal medicine complex extract improves arthritis, and completed the present invention.

[0009]

[0010] The purpose of the present invention is to provide a food composition for preventing or improving arthritis, comprising an extract of Ostericum KoreanumMaxim, an extract of Cibotium barometzJ. Smith, and an extract of safflower (Carthamus tinctoriusL.).

[0011] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating arthritis, comprising an extract of Ostericum KoreanumMaxim, an extract of Cibotium barometzJ. Smith, and an extract of Carthamus tinctoriusL.

[0012] Another object of the present invention is to provide an injection composition for preventing or treating arthritis, comprising an extract of Ostericum KoreanumMaxim, an extract of Cibotium barometzJ. Smith, and an extract of Carthamus tinctoriusL.

[0013] Another object of the present invention is to provide a method for preventing or treating arthritis, comprising administering to a subject a composition comprising an extract of Ostericum KoreanumMaxim, an extract of Cibotium barometzJ. Smith, and an extract of Carthamus tinctoriusL.

[0014]

[0015] In order to achieve the above purpose, the present invention provides a food composition for preventing or improving arthritis, comprising an extract of Ostericum Koreanum Maxim, an extract of Cibotium barometz J. Smith, and an extract of safflower (Carthamus tinctorius L.).

[0016] In addition, the present invention provides a pharmaceutical composition for preventing or treating arthritis, comprising an extract of Ostericum Koreanum Maxim, an extract of Cibotium barometz J. Smith, and an extract of Carthamus tinctorius L.

[0017] In addition, the present invention provides an injection composition for preventing or treating arthritis, comprising an extract of Ostericum Koreanum Maxim, an extract of Cibotium barometz J. Smith, and an extract of Carthamus tinctorius L.

[0018] In addition, the present invention provides a method for preventing or treating arthritis, comprising administering to a subject a composition comprising an extract of Ostericum KoreanumMaxim, an extract of Cibotium barometzJ. Smith, and an extract of Carthamus tinctoriusL.

[0019]

[0020] The herbal medicine complex extract of the present invention was confirmed to improve arthritis index and increase bone mineral mass and bone density in an animal model of osteoarthritis. Furthermore, it was confirmed to protect against cartilage damage, increasing cartilage thickness. Furthermore, it regulates the expression of tissue regeneration factors and inflammatory cytokines, making it useful in related industries.

[0021]

[0022] Figure 1 is a diagram illustrating a method for producing an animal model of osteoarthritis and administering a herbal medicine complex extract of the present invention.

[0023] Figure 2 is a diagram showing the administration location according to the administration cycle of the herbal medicine complex extract of the present invention.

[0024] Figures 3 and 4 are diagrams confirming the arthritis index in an animal model of osteoarthritis according to administration of the herbal medicine complex extract of the present invention.

[0025] Figures 5 and 6 are diagrams showing the inorganic bone mass and bone density in an animal model of osteoarthritis confirmed by X-ray absorptiometry according to administration of the herbal medicine complex extract of the present invention.

[0026] Figure 7 is a diagram showing the cartilage protection effect in an animal model of osteoarthritis following administration of the herbal medicine complex extract of the present invention, confirmed by H&E and toluidine blue staining.

[0027] Figures 8 and 9 are diagrams showing the cartilage protection effect of the herbal medicine complex extract of the present invention in an animal model of osteoarthritis confirmed by safranin O fast green staining.

[0028] Figures 10 and 11 are drawings showing the expression of MMP-1 confirmed by RT-PCR according to treatment with the herbal medicine complex extract of the present invention.

[0029] Figures 12 and 13 are drawings showing the expression of TNF-α confirmed by RT-PCR according to treatment with the herbal medicine complex extract of the present invention.

[0030] Figures 14 and 15 are drawings showing the expression of IL-6 confirmed by RT-PCR according to treatment with the herbal medicine complex extract of the present invention.

[0031]

[0032] 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.

[0033] 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.

[0034] The present invention provides a food composition for preventing or improving arthritis, comprising an extract of Ostericum Koreanum Maxim, an extract of Cibotium barometz J. Smith, and an extract of safflower (Carthamus tinctorius L.) as active ingredients.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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 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.

[0041] According to one embodiment of the present invention, the extract of Kang Hwal, the extract of Gu Cheok or the extract of Safflower may be extracted with a solvent selected from the group consisting of water, a lower alcohol of C1 to C4, a lower alcohol aqueous solution, hexane, chloroform and ethyl acetate.

[0042] According to one embodiment of the present invention, the extract of Kang Hwal, the extract of Gu Cheok, and the extract of Hong Hwa may be mixed in a weight ratio of 10:10:1.

[0043] According to one embodiment of the present invention, the composition may reduce an arthritis index.

[0044] According to one embodiment of the present invention, the composition may increase bone mineral content and bone mineral density.

[0045] According to one embodiment of the present invention, the composition may inhibit cartilage damage, and inhibiting cartilage damage may increase cartilage thickness.

[0046] According to one embodiment of the present invention, the composition may suppress the expression of a tissue regeneration factor and an inflammatory cytokine, wherein the tissue regeneration factor may be matrix metalloproteinase-1 (MMP-1), and the inflammatory cytokine may be tumor necrosis factor-α (TNF-α) or interleukin-6 (IL-6).

[0047] According to one embodiment of the present invention, the arthritis may be osteoarthritis.

[0048] In addition, the present invention provides a pharmaceutical composition for preventing or treating arthritis, which comprises an extract of Ostericum Koreanum Maxim, an extract of Cibotium barometz J. Smith, and an extract of Carthamus tinctorius L. as active ingredients.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] In addition, the present invention provides an injection composition for preventing or treating arthritis, which comprises an extract of Ostericum Koreanum Maxim, an extract of Cibotium barometz J. Smith, and an extract of Carthamus tinctorius L. as active ingredients.

[0057] The term "injection" used in the present invention refers to a method of administration that uses a syringe and a needle to inject a medicinal liquid into a part of the body for the purpose of local or systemic action, and can obtain the effect of the drug more accurately and quickly than conventional oral administration, suppository administration, or topical administration, and can effectively deliver the drug to the body even in cases where the drug cannot be administered orally, or in situations where the drug is denatured or difficult to absorb by digestive fluids. Injections are broadly classified into intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, and arterial injection. Intradermal injection is characterized by slow absorption and visible reactions, and is an injection method that can be performed for the diagnosis or prevention of diseases or to check for side effects of antibiotics before surgery. Subcutaneous injection is a method of administering a drug to the subcutaneous connective tissue, which is rapidly absorbed, and intramuscular injection is a method of injecting a drug into the muscle tissue of the buttocks or upper arm, and injecting the drug so that it is quickly absorbed by the high vascular distribution of the muscle. Intravenous injection, a method of administering medication directly into a vein, allows for rapid action and precise dosing. It is used in cases where intramuscular or subcutaneous injection is impractical due to the drug's irritating properties, allowing for the administration of large doses. Arterial injection, a method of injecting medication into an artery that flows into the affected area, allows for a direct effect on the affected area.

[0058] The above-mentioned injection refers to an aqueous, water-soluble, oily, suspension, turbid, or solid (administered after dissolution) formulation, and refers to a preparation formulated for injection purposes to allow the drug to act directly in the body without passing through the digestive tract. The conditions for an injection are as follows: 1) there are no excipients, 2) it is completely sterile, 3) it does not contain pyrogens, 4) it has an osmotic pressure similar to that of serum, 5) it has a pH similar to that of serum, and 6) it does not chemically react with the components of body tissues.

[0059] In addition, the above injections may contain solvents, solubilizers, buffers, isotonic agents, stabilizers, sulfating agents, analgesics, and suspending agents as additives according to the Ministry of Food and Drug Safety's guidelines for pharmaceutical additives. The "additives" that may be mixed into the injections are substances other than the active ingredients contained in the preparation, and are used for the purposes of increasing the usability of the drug, facilitating formulation, promoting stabilization of the preparation, and improving the appearance. As additives, excipients, stabilizers, preservatives, buffers, thickeners, suspending agents, emulsifiers, fragrances, solubilizers, colorants, and thickeners may be used as needed. However, the additives used mean that they do not exhibit direct pharmacological action in the dosage of the preparation, are safe, and do not change the therapeutic effect of the preparation or interfere with the test, which can be summarized as follows.

[0060] 1) Improved stability, bioavailability, etc.

[0061] 2) Maintaining the quality of the preparation during preservation or use

[0062] 3) Improving pharmacoeconomics by controlling the physical properties of drugs.

[0063] The "solvents" that can be mixed with the above additives include distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated Ringer's injection, ethanol, propylene glycol, non-volatile oils - sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, benzene benzoate, etc. "Solubilizing agents" that can be mixed include sodium benzoate, sodium salicylate, sodium acetate, urea, urethane monoethylacetamide, butazolidine, propylene glycol, Tween, nitrile acid amide, hexamine, dimethylacetamide, etc. "Buffering agents" may include weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptones, gums, etc. "Isotonic" agents may include sodium chloride, and "stabilizers" may include sodium bisulfite (NaHSO3), carbon dioxide gas, sodium metabisulfite (Na2S2O3), sodium sulfite (Na2SO3), nitrogen gas (N2), ethylenediaminetetraacetic acid, etc. "Oxidizing agents" may include sodium bisulfide 0.1%, sodium formaldehyde, sulfoxylate, thiourea, disodium ethylenediaminetetraacetate, acetone sodium bisulfite, etc. "Analgesic agents" may include benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, calcium gluconate, etc., and "suspending agents" may include sodium cisplatinum, sodium alginate, Tween 80, aluminum monostearate, etc.

[0064] In addition, the present invention provides a method for preventing or treating arthritis, comprising administering to a subject a composition comprising an extract of Ostericum Koreanum Maxim, an extract of Cibotium barometz J. Smith, and an extract of safflower (Carthamus tinctorius L.) as active ingredients.

[0065]

[0066] The advantages and features of the present invention, and the methods for achieving them, will become clearer with reference to the detailed examples described below. The present invention will now be described in detail through examples. However, these examples are intended to specifically illustrate the present invention and are not intended to limit the scope of the present invention.

[0067]

[0068] <Manufacturing Example 1> Manufacturing of herbal extracts

[0069] To prepare the herbal extract of the present invention, Ostericum Koreanum Maxim, Cibotium barometz J. Smith, and Carthaginium tinctorius L. were purchased from Dongyang Herb (Seoul, Korea). Ostericum Koreanum Maxim, Cibotium barometz J. Smith, and Carthaginium tinctorius L. were weighed in a weight ratio of 10:10:1, and then hot water extracted at 100°C for 2 hours using a multi-heating mantle. The extract was concentrated to 17.4 Brix % under reduced pressure using a rotary evaporator, and then powdered using a freeze dryer (yield: 19.43%). The powder of the composite extract was stored at 4°C until further analysis.

[0070]

[0071] <Experimental Example 1> Preparation for confirming the effect of herbal extracts on improving osteoarthritis

[0072] <1-1> Osteoarthritis animal model and local injection method of herbal medicine

[0073] Five-week-old male rats (Wistar rats) weighing 100 to 140 g were habituated for 7 days in an environment with a temperature of 22 ± 2℃ and a humidity of 50 ± 5% under a 12-hour light / dark cycle, with free access to food and water. Afterwards, 60 mg / ml of MIA (Monoiodoacetate, Sigma, SLBS5337) was injected into the knee joint to induce osteoarthritis. Afterwards, the complex extract of Manufacturing Example 1 was injected into the muscles around the knee synovium twice a week for 4 weeks for a total of 8 times. The concentration of the complex extract was prepared at 0.0940 (FLEXA_L group) or 0.9398 (FLEXA_H group) mg / ml, and 25 μl of the complex extract was administered in different directions to the periarticular region of the knee on both sides of the rats for each administration. The specific experimental schedule and injection sites are shown in Figures 1 and 2. The control groups used were the Normal group (NOR), the disease control group (MIA), and the positive control group (indomethacin, IDM).

[0074]

[0075] <1-2> Assessment of the severity of anteroposterior femoral arthritis

[0076] The arthritic rats of Experimental Example 1-1 were humanely sacrificed, and the femurs were separated from the tibias. Photographs of the femurs were taken, and five researchers, unaware of the experimental conditions, assessed the severity of arthritis using an established scoring system. The arthritis index was measured for inflammation and irregularities in each knee joint, and then summed to obtain a total arthritis score. The specific arthritis index measurement method is described below.

[0077] 0 points = normal

[0078] 1 point = mild redness and swelling

[0079] 2 points = significant redness and swelling of the knee joint

[0080] 3 points = Severe redness and swelling of the knee joint

[0081] 4 points = Severe redness and swelling of the entire knee joint

[0082] 5 points = highest knee joint inflammation

[0083] Arthritis severity was assessed using a validated scoring system, with parameters such as joint swelling, redness, and impaired mobility assessed after scoring. Statistical analysis was performed to determine the correlation between the arthritis index and experimental variables.

[0084]

[0085] <1-3> Evaluation of bone mineral mass and bone density

[0086] To measure bone mineral content (BMC) and bone mineral density (BMD) of the femurs and tibias obtained from the rats sacrificed in Experimental Examples 1-2 above, they were scanned using a dual-energy X-ray absorptiometer (DXA; InAlyzer dual X-ray absorptiometry, Medikors). After separating the images into bone and soft tissue components using the bone mode of DXA, the captured images were analyzed to quantify bone mineral content and bone density. Statistical analysis was performed using the GraphPad Prism 5 program, and the BMC and BMD values ​​were compared between the experimental groups and the intervention effects on bone health parameters were evaluated.

[0087]

[0088] <1-4> Histological evaluation of the posterior femur

[0089] The femur tissues obtained from the rats sacrificed in Experimental Example 1-2 above were fixed in 10% paraformaldehyde for 24 hours, washed, and the fixative that had penetrated the tissue was removed. Then, the tissues were decalcified by immersion in 0.5 M EDTA (Ethylenediaminetetraacetic Acid, MBcell, MB-E4422) for 7 weeks to remove minerals within the tissue. After decalcification, the tissues were dehydrated by immersion in alcohol concentrations of 70%, 80%, 90%, and 95% in that order, and paraffin blocks were made using xylene as a transparent agent. Paraffin blocks were sectioned at 10 μm intervals by microtome cutting, and after deparaffinization and hydration, stained with Hematoxylin & Eosin (H&E), Toluidine Blue, and Safranin O fast green, and histologically evaluated. The obtained optical microscope images were used to measure proteoglycan and mast cell diameters for bone tissue size analysis using the Image J program.

[0090]

[0091] <1-5> Measurement of tissue regeneration factors and inflammatory cytokine expression

[0092] The expression regulation effect of the herbal medicine complex extract of the present invention on tissue regeneration factors and inflammatory cytokines was confirmed. Specifically, human chondrosarcoma cell line SW1353 (ATCC, USA) was cultured in DMEM (Dulbecco's Modified Eagle Medium, Gibco, USA) containing 10% fetal bovine serum (FBS), 2 mM glutamine (Gibco, USA), 100 IU / ml penicillin (Gibco, USA), and 100 μg / ml streptomycin (Gibco, USA). The culture conditions were 5% CO2, 95% relative humidity, and 37°C. To confirm the effect of the complex extract, cells were seeded in 6-well plates at 2 X 10 5 The cells were seeded at a concentration of 10 μl and cultured for 24 hours with 100 μg / ml of extracts of Kangwhal, Gucheok, and Honghwa alone or in combination with 10 μl of recombinant interleukin 1β (IL-1β). After completion of the culture, RNA was extracted from SW1353 cells using TRIzol reagent (Invitrogen, USA), and cDNA was synthesized using a cDNA synthesis kit (Invitrogen, USA). The expression of tissue regeneration factors and inflammatory cytokines was confirmed using RT-PCR. The primer sequences used for RT-PCR are shown in Table 1. The NOR group, which is an untreated control group, the negative control group stimulated with IL-1β, and the group treated with Kangwhal, Gucheok, and Honghwa alone extracts were used as controls.

[0093] Gene namePrimer(5'-3')GAPDHFCCATCACCATCTTCCAGGAGRCCTGCTTCACCACCTTCTTGMMP-1FGCTGGGAGCAAACACATCTGAGGTRTGAGCCGCAACACGATGTAAGTTGTNF-αFTCTACTCCCAGGTCCTCTTCRAAGTAGACCTGCCCAGACTCIL-6FATGAACTCCTTCTCCACAAGCGCRGAAGAGCCCTCAGGCTGGACTG

[0094]

[0095] <1-6> Statistical analysis

[0096] Statistical analysis was performed using GraphPad Prism 5 (GraphPad Software Inc., USA). Data are presented as the mean ± standard deviation (SD) from at least three independent experiments. Statistical differences between groups were assessed using one-way analysis of variance (ANOVA) followed by Tukey's test. p<0.05 was considered statistically significant.

[0097]

[0098] <Example 1> Confirmation of improvement in cartilage and arthritis indices of the composite extract

[0099] In MIA-induced osteoarthritis, it was confirmed whether the complex extract of the present invention improves the cartilage and arthritis index within the joint cavity. As a result, as shown in Figures 3 and 4, the NOR group showed a smooth and undamaged surface with a glossy appearance in the form of a normal knee joint, whereas the MIA group showed cartilage erosion in both the anterior and posterior femurs. In the FLEXA_H group, which was administered a high concentration of the complex extract, it was confirmed that damage to the articular cartilage structure was suppressed. In addition, the arthritis index of the anterior femur was 5.93 times higher in the MIA group compared to the NOR group, and the arthritis index was confirmed to be recovered by 5.87% in the complex extract FLEXA_L group and 52.56% in the FLEXA_H group compared to the MIA-induced group. In the posterior femur, the MIA group showed a 5.92-fold increase in the arthritis index compared to the NOR group, and a 57.08% recovery was confirmed in the FLEXA_H group, confirming that the herbal medicine complex extract of the present invention protects articular cartilage and improves the arthritis index.

[0100]

[0101] <Example 2> Confirmation of increased bone mineral mass and bone density of the composite extract

[0102] It was confirmed whether the complex extract of the present invention increases bone mineral mass and bone density in osteoarthritis induced by MIA. As a result, as shown in Figures 5 and 6, compared to the NOR group, bone mineral mass was decreased by 36.9% in the MIA group, but the decreased bone mineral mass was confirmed to increase by 11.1% when administered a low concentration of the complex extract and by 35.7% when administered a high concentration of the complex extract. In addition, bone density was confirmed to decrease by 50.7% in the MIA group compared to the NOR group, but the decreased bone density was confirmed to increase by 3.9% in the FLEXA_L group and 56.6% in the FLEXA_H group.

[0103]

[0104] <Example 3> Confirmation of the cartilage protection effect of the complex extract

[0105] In the case of osteoarthritis induced by MIA, the composite extract of the present invention was confirmed to protect cartilage tissue. As a result, as shown in Fig. 7, in the MIA group, the population of chondrocytes in the cartilage was reduced, and the density of chondrocytes was confirmed to be reduced in the area affected by osteoarthritis. In addition, in the MIA group, the population of adipocytes increased due to the induction of osteoarthritis, and it was confirmed that proteoglycan, the main glycoprotein of cartilage, was reduced (Figs. 8 and 9). In the MIA group, the articular cartilage of the subchondral plate was significantly thinned, and compared to the NOR group, the articular cartilage of the subchondral plate was reduced by 4.32 times. However, the articular cartilage of the reduced subchondral plate was recovered by 1.72 times in the FLEXA_L group, and the thickness of the cartilage was recovered by approximately 4.19 times in the FLEXA_H group, confirming that the herbal composite extract of the present invention protects cartilage.

[0106]

[0107] <Example 4> Confirmation of tissue regeneration and inflammatory cytokine regulation of complex extracts

[0108] It was confirmed whether the complex extract of the present invention regulates tissue regeneration factors and inflammatory cytokines. Specifically, matrix metalloproteinase-1 (MMP-1) is a collagen-degrading enzyme that promotes collagen degradation in cartilage tissue, inflammatory responses that affect the progression of osteoarthritis, and contributes to structural damage and restructuring of cartilage tissue. Therefore, the expression of MMP-1 was confirmed. As a result, as shown in Figures 10 and 11, compared to the control group, the expression level of MMP-1 in the group stimulated with IL-1β significantly increased to 10.64 ± 0.12. However, the expression level in the group administered with Kangwhal extract was 6.18 ± 0.10, the group administered with Gucheok extract was 6.62 ± 0.12, and the group administered with Honghwa extract was 9.12 ± 0.05. It was confirmed that the expression level of MMP-1 was significantly reduced to 2.88 ± 0.08 in the group administered with the complex extract. Comparing the MMP-1 expression levels of each group, the MMP-1 expression level of the complex (Formula) was reduced by 72.92%, confirming the synergistic effect of the complex extract.

[0109] In addition, when the expression of inflammatory cytokines TNF-α (tumor necrosis factor-α) and IL-6 (Interleukin-6) was confirmed, the expression level of TNF-α in the group stimulated with IL-1β significantly increased to 3.58 ± 0.19 compared to the control group. The expression level of TNF-α in the group administered with Kangwhal extract was 1.97 ± 0.00, the expression level in the group administered with Gucheok extract was 2.34 ± 0.07, and the expression level in the group administered with Honghwa extract was 1.86 ± 0.04. In the group administered with the complex extract, the expression level of TNF-α was confirmed to have significantly decreased to 1.46 ± 0.02. When comparing the expression levels of TNF-α in each group, the group administered the complex extract showed a 59.17% decrease in TNF-α expression levels, confirming the synergistic effect of the complex extract (Figs. 12 and 13).

[0110] In addition, compared to the control group, the expression level of IL-6 in the group stimulated with IL-1β significantly increased to 9.47 ± 0.26. The expression level of IL-6 in the group administered with the extract of Kangwhal was 7.10 ± 0.21, the expression level of the group administered with the extract of Gucheok was 6.98 ± 0.06, and the expression level of the group administered with the extract of Honghwa was 3.35 ± 0.14. In the group administered with the complex extract, the expression level of IL-6 was confirmed to significantly decrease to 3.11 ± 0.05. Comparing the expression level of IL-6 in each group, the group administered with the complex extract showed a 67.17% decrease in the expression level of IL-6 (Figs. 14 and 15).

[0111]

[0112] Therefore, the herbal medicine complex extract of the present invention was confirmed to improve arthritis index and increase bone mineral mass and bone density in an animal model of osteoarthritis. Furthermore, it was confirmed to protect against cartilage damage, increasing cartilage thickness, and regulating the expression of tissue regeneration factors and inflammatory cytokines.

Claims

1. A food composition for preventing or improving arthritis, comprising an extract of Ostericum Koreanum Maxim, an extract of Cibotium barometz J. Smith, and an extract of Carthamus tinctorius L.

2. A composition according to claim 1, wherein the extract of the strong root is extracted with a solvent selected from the group consisting of water, lower alcohols of C1 to C4, lower alcohol aqueous solutions, hexane, chloroform, and ethyl acetate.

3. A composition in the first paragraph, wherein the extract is extracted with a solvent selected from the group consisting of water, lower alcohols of C1 to C4, lower alcohol aqueous solutions, hexane, chloroform, and ethyl acetate.

4. A composition according to claim 1, wherein the safflower extract is extracted with a solvent selected from the group consisting of water, lower alcohols of C1 to C4, lower alcohol aqueous solutions, hexane, chloroform, and ethyl acetate.

5. A composition in the first paragraph, wherein the extract of Gangwhal, the extract of Gucheok, and the extract of Honghwa are mixed in a weight ratio of 10:10:

1.

6. A composition according to claim 1, wherein the composition reduces an arthritis index.

7. A composition according to claim 1, wherein the composition increases bone mineral content and bone mineral density.

8. A composition according to claim 1, wherein the composition inhibits cartilage damage.

9. A composition according to claim 8, wherein the suppression of cartilage damage is achieved by increasing cartilage thickness.

10. A composition according to claim 1, wherein the composition inhibits the expression of tissue regeneration factors and inflammatory cytokines.

11. A composition according to claim 10, wherein the tissue regeneration factor is matrix metalloproteinase-1 (MMP-1).

12. A composition according to claim 10, wherein the inflammatory cytokine is TNF-α (tumor necrosis factor-α) or IL-6 (Interleukin-6).

13. A composition according to claim 1, wherein the arthritis is osteoarthritis.

14. A pharmaceutical composition for preventing or treating arthritis, comprising an extract of Ostericum Koreanum Maxim, an extract of Cibotium barometz J. Smith, and an extract of Carthamus tinctorius L.

15. An injection composition for preventing or treating arthritis comprising an extract of Ostericum Koreanum Maxim, an extract of Cibotium barometz J. Smith, and an extract of Carthamus tinctorius L.

16. A method for preventing or treating arthritis, comprising administering to a subject a composition comprising an extract of Ostericum KoreanumMaxim, an extract of Cibotium barometzJ. Smith, and an extract of safflower (Carthamus tinctoriusL.).

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