KR102143244B1 - Composition for preventing, improving or treating arthritis comprising extract of Ganoderma lucidum, extract of Artemisia princeps or mixture thereof - Google Patents

Abstract

The present invention relates to a composition for the prevention, improvement or treatment of arthritis, comprising an extract of Psyllium Ganoderma lucidum extract, a wormwood extract or a mixture thereof as an active ingredient, and more particularly, of Mmp3 or Mmp13 increased by IL-1β in chondrocytes. A pharmaceutical composition for preventing or treating arthritis and/or a pharmaceutical composition for preventing or improving arthritis, comprising as an active ingredient a skeletal ganoderma lucidum extract, wormwood extract, or a mixture thereof, which exhibits the effect of suppressing joint inflammation or cartilage destruction by reducing mRNA or protein expression It provides a health functional food composition for use.

Description

Composition for preventing, improving or treating arthritis comprising extract of Ganoderma lucidum, extract of Artemisia princeps or mixture thereof, comprising as an active ingredient

The present invention relates to a composition for the prevention, improvement or treatment of arthritis, comprising an extract of Psyllium Ganoderma lucidum extract, a wormwood extract or a mixture thereof as an active ingredient, and more particularly, of Mmp3 or Mmp13 increased by IL-1β in chondrocytes. A pharmaceutical composition for preventing or treating arthritis and/or a pharmaceutical composition for preventing or improving arthritis, comprising as an active ingredient a skeletal ganoderma lucidum extract, wormwood extract, or a mixture thereof, which exhibits the effect of suppressing joint inflammation or cartilage destruction by reducing mRNA or protein expression It provides a health functional food composition for use.

Arthritis is largely divided into degenerative arthritis and rheumatoid arthritis. Degenerative arthritis is a disease that occurs mainly in middle or old age, and causes pain, stiffness and movement disorders in joints that receive a lot of weight, such as knee joints and hip joints. In the past, it was simply thought of as an aging phenomenon, but now it is thought that symptoms appear differently due to a combination of various causes such as age, genetic factors, obesity, joint shape, and hormones.

It is known that degenerative arthritis mainly involves the formation of chronic inflammation due to the gradual damage to the articular cartilage, and thus a process of degeneration of surrounding tissues. More specifically, when chronic inflammation occurs as transcription factors such as NF-kB and AP-1 are activated in cartilage tissue by accumulation of physical stress, factors that mediate and amplify the inflammatory response such as COX-2 and LOX are expressed. And the surrounding blood circulation is suppressed, and tissue destruction factors such as matrix metalloproteinases (MMPs), hyaluronidase, and iNOS are overexpressed to destroy cartilage tissue. , Tendons, ligaments, and other tissues are also affected, causing severe pain.

Rheumatoid arthritis is an inflammatory autoimmune disease that occurs in multiple joints. In the synovial tissue and synovial fluid of patients with rheumatoid arthritis symptoms, inflammatory cells (macrophages, T-cells, B cells, dendritic cells, etc.) act excessively, causing chronic inflammation, causing joint and cartilage damage, and pain. It is characterized by causing.

Until now, no treatment for arthritis has been developed, and non-steroidal anti-inflammatory drugs (NSAIDs) are generally used to relieve joint inflammation. However, since the NSAID series drugs have the main purpose of temporarily alleviating joint inflammation, the use of NSAID drugs is not suitable for degenerative arthritis, which is a non-inflammatory arthritis that requires promotion of cartilage formation and suppression of cartilage tissue destruction. Pritchard MH et al., Annals of the Rheumatic Diseases, 37:493-503, 1978). These nonsteroidal anti-inflammatory drugs (NSAIDs) are suitable for blocking the inflammatory mechanism as a therapeutic agent for rheumatoid arthritis, an inflammatory arthritis, but rather accelerate cartilage damage or have side effects such as cardiovascular, gastrointestinal, kidney, and liver.

In addition, the self-derived chondrocyte transplantation, which is currently developed for cartilage formation, is a method of generating supernatural bone by collecting cartilage and subchondral parts already generated from the normal part of the patient, digging an appropriate hole on the damaged cartilage part, and transplanting it. Although it has been successful in some patients, it cannot be a universal method as it can only be performed on patients with less cartilage tissue destruction and capable of autografting (Peterson L et al., J Bone Joint Surg Am. 85-A Suppl :17-24, 2003).

Therefore, the development of new drugs for the prevention, improvement or treatment of arthritis is still required, and among them, studies using natural products with little side effects are being conducted. Korean Patent Application Publication No. 10-2017-0111272 discloses a cosmetic composition comprising a fermented extract of reishi mushrooms, and Korean Patent Publication No. 10-2018-0074296 discloses a chocolate for preventing and improving obesity including a reishi mushroom extract and its It discloses a manufacturing method, and Korean Patent Registration No. 10-1286133 discloses a skin cosmetic composition having a deodorant and antibacterial effect comprising Ganghwa Lion Balsam and persimmon leaf extract as active ingredients, and Korean Patent Publication No. 10-2009- No. 0064700 discloses a pharmaceutical composition for the prevention or treatment of cancer diseases or cardiac circulatory diseases, comprising as an active ingredient, Eupafoline extracted from Mugwort. However, there is no known effect on the treatment of arthritis with the extracts of Pyeonggak Reishi mushroom, Mugwort Mugwort extract, or mixtures thereof.

The inventors of the present invention by confirming that it can suppress joint inflammation and cartilage destruction by reducing the mRNA or protein expression level of Mmp3 or Mmp13 increased by IL-1β in chondrocytes, by confirming that the P. The present invention has been completed.

It is an object of the present invention to provide a composition for preventing, improving or treating arthritis using Ganoderma lucidum extract, Artemisia princeps extract or a mixture thereof.

In order to solve the above-described problem, the present invention provides a pharmaceutical composition for preventing or treating arthritis, comprising an extract of Ganoderma lucidum , an Artemisia princeps extract or a mixture thereof as an active ingredient.

In addition, the present invention, Ganoderma lucidum ) extract, artemisia princeps ) extract or a mixture thereof as an active ingredient to provide a health functional food composition for preventing or improving arthritis.

According to a preferred embodiment of the present invention, the extract of Pyeonggak reishi mushroom is extracted from all parts except the medium of Pyeonggak Gyeongji mushroom, and the extract of Mugwort may be extracted from the above-ground part of Mugwort.

According to a preferred embodiment of the present invention, the Prunus Ganoderma lucidum extract or Mugwort Mugwort extract is extracted using water, C ₁ to C ₄ lower alcohol or a mixture thereof as a solvent, and the mixture is It may be a mixture of wormwood extract.

According to another preferred embodiment of the present invention, the mixture may be obtained by mixing the Pyeonggak Reishi mushroom extract and the Mugwort Mugwort extract in a weight ratio of 1:1 to 1:20.

According to another preferred embodiment of the present invention, the arthritis may be degenerative arthritis or rheumatoid arthritis.

According to another preferred embodiment of the present invention, the composition may suppress joint inflammation or cartilage destruction by reducing the mRNA or protein expression of Mmp3 or Mmp-13.

Ganoderma ( Ganoderma) of the present invention lucidum ) extract, Artemisia princeps extract, or a mixture thereof as an active ingredient, by reducing the mRNA or protein expression of Mmp3 or Mmp-13 increased by IL-1β in chondrocytes, joint inflammation or cartilage destruction It shows the effect of suppressing. Accordingly, the composition of the present invention is useful for the prevention, amelioration or treatment of arthritis, particularly degenerative arthritis.

1 is a result of confirming that the Mugwort Mugwort extract is treated at different concentrations (0, 10, 50, 100 or 200 μg/ml) to chondrocytes, and the Mugwort Mugwort extract does not show cytotoxicity to the chondrocytes.
Figure 2 is a result of confirming that the Pyeongak Ganoderma lucidum extract is treated at different concentrations (0, 5, 10 or 50 μg/ml) to the chondrocytes, and the Prunus Ganoderma lucidum extract does not show cytotoxicity to the chondrocytes.
3A and 3B show the results of confirming that the expression of mRNA and protein of Mmp3 and Mmp13, which induce joint destruction in chondrocytes, is increased by IL-1β, but is decreased in a concentration-dependent manner by the extract of Mugwort.
FIG. 4 is a result of confirming that the increased mRNA expression of Mmp3 and Mmp13, which induces joint destruction in chondrocytes, is increased by IL-1β, but is decreased in a concentration-dependent manner by the extract of deciduous ganoderma lucidum.
Figure 5 is a result of confirming that the mRNA expression of Mmp3 and Mmp13, which induces joint destruction in chondrocytes, is increased by IL-1β, but is significantly reduced by a mixture of a wormwood extract or a mixture of wormwood extract and an extract .

As described above, conventionally, non-steroidal anti-inflammatory drugs (NSAIDs) have been used for the purpose of relieving joint inflammation, but NSAID-based drugs have the main purpose of temporarily alleviating joint inflammation, so that it promotes cartilage formation and promotes cartilage. It is not suitable for degenerative arthritis, which is a non-inflammatory arthritis that requires tissue destruction, and the autologous chondrocyte transplantation developed for cartilage formation involves collecting the cartilage and subchondral parts already generated in the normal part of the patient together, and the damaged cartilage part. The method of generating supernatural bone by digging a suitable hole in the top and transplanting has been successful in some patients, but there was a problem that it could not be a universal method because it can only be performed on patients with less cartilage tissue destruction and capable of autografting. .

The present inventors have sought a solution to the above-described problem by providing a composition for preventing, improving or treating arthritis using a prune ganoderma lucidum extract, a wormwood extract or a mixture thereof. The composition comprising the P. ganoderma lucidum extract, Mugwort extract or a mixture thereof according to the present invention as an active ingredient destroys joint inflammation and cartilage by reducing the mRNA or protein expression level of Mmp3 or Mmp13 increased by IL-1β in chondrocytes. Can be suppressed. Accordingly, the composition of the present invention is useful for preventing, ameliorating or treating arthritis, particularly degenerative arthritis.

Terms used in the present invention are defined as follows.

The term “pharmaceutical composition” refers to a mixture of Ganoderma lucidum extract, Artemisia princeps extract or mixtures thereof with other chemical components such as a diluent or carrier.

The term “carrier” is defined as a compound that facilitates the addition of the compound into a cell or tissue. For example, dimethyl sulfoxide (DMSO) is a commonly used carrier that facilitates the introduction of many organic compounds into a cell or tissue of an organism.

The term “diluent” is defined as a compound that is diluted in water that will dissolve the compound as well as stabilize the biologically active form of the subject compound. Salts dissolved in buffer solutions are used as diluents in the art. A commonly used buffer solution is phosphate buffered saline, because it mimics the salt state of human solutions. Because buffer salts can control the pH of the solution at low concentrations, buffer diluents rarely modify the biological activity of the compound.

The term “treatment” refers to an approach to obtaining beneficial or desirable clinical outcomes. For the purposes of the present invention, beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, reduction of disease range, stabilization of disease state (i.e., not exacerbation), delay or decrease in disease progression, disease state. Amelioration or temporal alleviation and alleviation of (partially or entirely), detectable or undetectable. In addition, “treatment” may mean increasing the survival rate compared to the expected survival rate when not receiving treatment. “Treatment” refers to both therapeutic treatment and prophylactic or preventative measures. These treatments include the disorder to be prevented as well as the treatment required for a disorder that has already occurred. “Alleviating” the disease is a reduction in the extent of the condition and/or undesirable clinical signs and/or a delayed or prolonged time course of progression compared to untreated. Means to lose.

“Synergistic” means that the effect that occurs when each component is administered in combination (combination) is greater than the sum of the effects that occur when administered alone as a single component [Chou and Talalay, Adv. Enzyme. Regul., 22:27-55, 1984]. The mixture of the extract of P. ganoderma lucidum and Mugwort extract of the present invention exhibits a synergistic effect in reducing the mRNA or protein expression of Mmp3 or Mmp13.

All technical terms used in the present invention, unless defined otherwise, are used in the sense as commonly understood by those skilled in the art in the relevant field of the present invention. In addition, although preferred methods or samples are described in the present specification, those similar or equivalent are included in the scope of the present invention.

Hereinafter, the present invention will be described in more detail.

The present invention is Ganoderma ( Ganoderma) lucidum ) extract, Artemisia princeps ) provides a pharmaceutical composition for the prevention or treatment of arthritis comprising an extract or a mixture thereof as an active ingredient.

Ganoderma lucidum (Fr) is a mushroom that has been used as a rare medicinal material in Korea, China, and Japan, and is widely distributed worldwide in tropical, subtropical and temperate regions. says karst. In addition to polysaccharides, reishi mushrooms contain various substances such as triterpenes, nucleosides, steroids, fatty acids, alkaloids, proteins, amino acids, and inorganic salts, among which high molecular substances (polysaccharides, etc.) and low molecular substances (triterpenes, etc.) are various. Do. Low-molecular substances have anti-inflammatory, antioxidant, hepatocellular protection, anti-hypertension, cholesterol lowering, and platelet aggregation inhibition, and high molecular substances have reported effects such as lowering blood pressure, blood stasis, improving hyperlipidemia, lowering blood sugar, immunity, and anti-tumor. Became. In particular, the main secondary metabolites having physiological activity and pharmacological function of Ganoderma lucidum are polysaccharides and triterpenes, and various medicinal effects and active ingredients related to these substances have been scientifically identified.

Ganoderma lucidum mushrooms are largely divided into flat and green squares. Ganodermalucidum is the same as Ganodermalucidum, but the physiologically active substances and physiological efficacy are different. Antler-shaped Ganoderma lucidum is a type of Ganoderma lucidum mushroom that grows naturally on the stump of a broad-leaved tree in the shape of an antler shape. It is not easily found in nature and is highly evaluated medically in China and Japan. In Korea, nogak ganoderma lucidum has a high yield but poor marketability, so it has been cultivated a lot in the early 1980s, the early stage of cultivation of ganoderma lucidum, but has not been cultivated thereafter, but is recently cultivated in some farms for ornamental and processed products. However, it has recently been reported that P. ganoderma lucidum mushrooms contain higher amounts of beta-D-glucan and triterpenoids than prunus ganoderma lucidum mushrooms, and have a stronger physiological activity in enhancing immunity and suppressing tumors. Kidney-shaped Ganoderma lucidum refers to Ganoderma lucidum mushrooms that grow naturally on broad-leaved tree stumps that have a flat shape without a sack. Generally, Ganoderma lucidum on the market are Pyeonggak Ganoderma mushrooms. The pharmaceutical composition of the present invention was used in all parts except the medium of Pyeonggak Reishi mushroom.

Artemisia princeps Pampanini (Artemisia princeps Pampanini) is a mugwort belonging to the Artemisia species, and it is named because it looks similar to lion's feet. Artemisia princeps Pampanini (Artemisia princeps Pampanini) is a perennial herb that grows naturally only in Ganghwa-do, and has been widely used as an anti-inflammatory, analgesic, cardiac, antitussive, and inhalant in oriental medicine. It is known for its antibacterial action as its main pharmacological action. The wormwood extract used in the present invention may be prepared from the wormwood outpost as a raw material, and preferably may be prepared from the above-ground parts including stems and leaves as a raw material.

Pyeongak gyeongji mushrooms and wormwood wormwood used in the present invention may be purchased and used commercially, or may be directly collected or cultivated in nature.

The term "extract" of the present invention refers to an extract obtained by the extraction treatment of the P. ganoderma lucidum or Mugwort, a diluted solution or a pesticide solution of the extract, a dried product obtained by drying the extract, a prepared product or purified product of the extract, or these It includes the extract of all formulations that can be formed using the extract itself and the extract, such as a mixture of the extract. Specifically, the extract of the present invention may be prepared and used in a dry powder form after extraction.

In the present invention, the kind of the extraction solvent used to extract the P. ganoderma lucidum and Mugwort is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the extraction solvent include water; C ₁ to C ₄ lower alcohols such as methanol, ethanol, propyl alcohol and butyl alcohol; Polyhydric alcohols such as glycerin, butylene glycol, and propylene glycol; And hydrocarbon-based solvents such as methyl acetate, ethyl acetate, acetone, benzene, hexane, diethyl ether, and dichloromethane; Alternatively, a mixture thereof may be used, and water, a C ₁ to C ₄ lower alcohol or a mixture thereof is preferably used as a solvent, and the lower alcohol is ethanol (alcohol), methanol or butanol.

It is preferable that the Pyeonggak Ganoderma lucidum extract and Mugwort Mugwort extract are each prepared by a manufacturing method comprising the following steps, but are not limited thereto:

1) extracting by adding an extraction solvent to Pyeongak Reishi Mushroom or Mugwort;

2) filtering the extract of step 1); And

3) A step of drying after concentrating the filtered extract of step 2) under reduced pressure.

In the above method, as the extraction method in step 1), conventional methods in the art such as filtration, hot water extraction, immersion extraction, reflux cooling extraction, and ultrasonic extraction may be used. The extraction process may be performed at, for example, 50°C to 150°C, or 75°C to 120°C, or 90°C to 110°C, but is not limited thereto. In addition, the extraction time is not particularly limited, but may be 10 minutes to 12 hours, or 30 minutes to 6 hours, or 2 hours to 4 hours.

In the above method, the vacuum concentration in step 3) is preferably a vacuum vacuum concentrator or a vacuum rotary evaporator, but is not limited thereto. In addition, the drying is preferably vacuum drying, vacuum drying, boiling drying, spray drying, or freeze drying, but is not limited thereto.

In the pharmaceutical composition of the present invention, the mixture is a mixture of a single extract of Pyeonggak Ganoderma lucidum and a single extract of Mugwort sprouting, and the extract of Pyeonggak Gyeongji and Mugwort extract may be mixed in a weight ratio of 1:1 to 1:20, , Preferably 1:2 to 1:10, more preferably 1:2 or more and less than 1:10 may be mixed in a weight ratio. When mixing at a weight ratio outside the above range, the synergistic effect of the mixture decreases, and the optimum activity cannot be exhibited.

In the pharmaceutical composition of the present invention, the arthritis may be degenerative arthritis or rheumatoid arthritis, preferably degenerative arthritis.

The degenerative arthritis refers to arthritis caused by degenerative changes in cartilage and surrounding bones in a synovial joint. In other words, degenerative arthritis is characterized by gradual loss of articular cartilage, as well as enlargement of bone located below the cartilage, bone formation at the edge of the joint, and non-specific synovial inflammation, and aging or excessive physical pressure (e.g., obesity, It is a disease caused by damage to cartilage due to trauma). Therefore, degenerative arthritis shows severe pain and movement disorders in joints that receive a lot of weight, ie, knee (knee) joints, hip (hip) joints, etc., and when left unattended for a long period of time, even joint deformation is caused.

Mmp3 or Mmp13 mRNA increased by IL-1β, a cytokine associated with degenerative arthritis, as shown in Figs. 3 to 5, or It inhibits joint inflammation or cartilage destruction by reducing protein expression. Particularly, a mixture of P. ganoderma lucidum extract and Mugwort Mugwort extract at a specific weight ratio exhibits a synergy effect on reducing the expression of Mmp3 or Mmp13 mRNA or protein. Therefore, the pharmaceutical composition of the present invention is useful for preventing, ameliorating or treating degenerative arthritis induced by IL-1β.

The pharmaceutical composition of the present invention may further include suitable carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions.

The pharmaceutical composition according to the present invention is formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injectable solutions, respectively, according to a conventional method. Can be used.

Carriers, excipients, and diluents that may be contained in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate. , Gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils.

When formulating the pharmaceutical composition of the present invention, it is prepared using diluents or excipients such as generally used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient for the compound, such as starch, calcium carbonate, sucrose or lactose. , Gelatin, etc. are mixed to prepare it. In addition, lubricants such as magnesium stearate and talc are used in addition to simple excipients. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, and preservatives may be included. .

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount.

In the present invention, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type and severity of the individual, age, sex, progression of the disease The degree, activity of the drug, sensitivity to the drug, time of administration, route of administration and rate of excretion, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And it can be administered single or multiple.

The composition may be administered to mammals such as mice, mice, livestock, and humans by various routes. All modes of administration can be expected and can be administered, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injection.

In addition, the present invention, Ganoderma lucidum ) extract, Artemisia princeps ) It provides a health functional food composition for preventing or improving arthritis comprising an extract or a mixture thereof as an active ingredient.

In the health functional food composition, since the description of the Pyeonggak Reishi mushroom extract, the Mugwort Mugwort extract or a mixture thereof is the same as described above, the description thereof will be omitted to avoid excessive complexity of the present specification due to redundant description.

Likewise, since the description of arthritis in the health functional food composition is the same as described above, the description thereof will be omitted in order to avoid excessive complexity of the present specification due to redundant description.

In the present invention, the term "health functional food" refers to a food manufactured and processed using raw materials or ingredients having useful functions for the human body according to the Health Functional Food Act No.6727, and nutrients for the structure and function of the human body It means ingestion for the purpose of controlling or obtaining useful effects for health purposes such as physiological effects.

The health functional food of the present invention may contain ordinary food additives, and whether it is suitable as a food additive is determined according to the general rules and general test methods of food additives approved by the Food and Drug Administration, unless otherwise specified. It is judged according to the standards and standards.

Examples of the items listed in the "Food Additives Code" include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as reduced pigment, licorice extract, crystalline cellulose, high color pigment, and guar gum; Mixed preparations, such as a sodium L-glutamate preparation, an alkali additive for noodles, a preservative preparation, and a tar color preparation, etc. are mentioned.

The health functional food composition of the present invention may contain various flavoring agents or natural carbohydrates as an additional component, like a conventional food composition.

Examples of the above-described natural carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, etc.; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. The above-described flavoring agent may advantageously be used a natural flavoring agent (taumatin), a stevia extract (eg, rebaudioside A, glycyrrhizin, etc.) and a synthetic flavoring agent (saccharin, aspartame, etc.).

The health functional food composition of the present invention may 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, etc. There is this.

For example, in the health functional food in the form of a tablet, a mixture obtained by mixing the active ingredient of the present invention with an excipient, a binder, a disintegrant, and other additives is granulated by a conventional method, and then a lubricant, etc. The mixture can be directly compression molded. In addition, the health functional food in the form of a tablet may contain a mating agent or the like if necessary.

Among the health functional foods in the form of capsules, hard capsules can be prepared by filling a conventional hard capsule with a mixture of the extract, which is the active ingredient of the present invention, with additives such as excipients, and the soft capsules contain the active ingredient with additives such as excipients. The mixed mixture can be prepared by filling a capsule base such as gelatin. The soft capsules may contain plasticizers such as glycerin or sorbitol, colorants, preservatives, and the like, if necessary.

The cyclic health functional food can be prepared by molding a mixture of the active ingredient of the present invention, a mixture of herbal extracts, excipients, binders, disintegrants, etc., by conventionally known methods. It can be stripped, or the surface can be coated with a material such as starch or talc.

The health functional food in the form of granules can be prepared in granular form by a mixture of the mixed herbal medicine extract, the active ingredient of the present invention, excipients, binders, disintegrants, etc., by a conventionally known method, and if necessary, flavoring agents and flavoring agents. And the like.

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

Preparation of Mugwort Mugwort and Pyeongak Reishi Mushroom Extract

1-1. Preparation of Mugwort Mugwort Extract

Purchasing a commercially available Pyeonggak Reishi Mushroom was powdered, except for the medium, and then statically extracted indoors for 24 hours with water (repeat twice), filtered with filter paper, and freeze-dried with a freeze dryer to powder. .

1-2. Preparation of Pyeongak Reishi Mushroom Extract

Commercially available Mugwort Mugwort was purchased, and the above-ground part was subjected to static extraction (repeat twice) indoors for 24 hours using alcohol, filtered through filter paper, concentrated to 80% with a concentrator, freeze-dried with a freeze dryer, and powdered.

Measurement of Chondrocyte Toxicity by Extracts of Mugwort or Prunus Ganoderma lucidum

2-1. Measurement of Chondrocyte Toxicity by Mugwort Extract

Chondrocytes were obtained from cartilage tissue derived from femoral heads, femoral condyles, and tibial plateaus of a 5-day-old normal mouse (Central Experimental Animal Co., Ltd.). The obtained chondrocytes were 10% (v/v) fetal bovine serum (Gibco, USA), 50 μg/ml of streptomycin (Sigma-Aldrich, USA) and 50 unit/ml penicillin (Sigma-Aldrich, USA). ) Was cultured in DMEM medium (Gibco, USA) containing.

To confirm that the wormwood extract does not show toxicity to chondrocytes, chondrocytes were cultured in a 96-well culture vessel on a 9×10 ³ cell/well basis, and then the wormwood extract was 0, 10, 50, 100 and 200. Each treatment was performed at a concentration of μg/ml and incubated in an incubator at 37° C. and 5% CO ₂ for 24 hours. Toxicity to chondrocytes of wormwood extract was confirmed by measuring absorbance at 450 nm using an EZ-Cytox cell survival assay kit (Dozen, Korea).

As a result, as shown in FIG. 1, it was confirmed that the extract of wormwood wormwood did not show cytotoxicity to chondrocytes at all concentrations of 10, 50 and 100 μg/ml, and did not negatively affect chondrocyte proliferation.

2-2. Measurement of Chondrocyte Toxicity by Extract of Prunus Ganoderma lucidum Mushroom

Chondrocyte toxicity was measured in the same manner as in Example 2-1, except that the concentrations of the chondrocytes treated P. ganoderma lucidum extract were 5, 10, and 50 μg/ml, respectively.

As shown in FIG. 2, it was confirmed that the P. ganoderma lucidum extract did not show cytotoxicity to chondrocytes at all concentrations of 5, 10 and 50 μg/ml, and did not negatively affect chondrocyte proliferation.

Inhibition of joint inflammation and cartilage destruction by Mugwort extract

3-1. Confirmation of the inhibitory effect of Mmp3 and Mmp13 on transcription levels

IL-1β is a representative inflammatory cytokine that promotes joint inflammation and cartilage tissue destruction in chondrocytes. In order to confirm the effect of inhibiting joint inflammation and cartilage destruction, the chondrocytes obtained in Example 2-1 were treated with 1 ng/ml of IL-1β (GeneScript, USA) and cultured for 36 hours, and then, the extract of Mugwort Each was treated with 0, 10, 50, and 100 μg/ml and cultured for an additional 24 hours. Chondrocytes not treated with anything were used as a control (Con).

Then, to perform qRT-PCR, RNA was extracted from chondrocytes using TRI reagent (Molecular Research Center Inc.), and the cDNA obtained by reverse transcription of the RNA was used with primers of SEQ ID NOs: 1, 2, 3 and 4 Then, the expression of Mmp3 and Mmp13, which were important for cartilage destruction, was confirmed by amplification by PCR under the annealing temperature of 58°C. As a control, Gapdh (450bp, annealing temperature 58°C) was confirmed with the primers of SEQ ID NOs: 5 and 6.

Sequence number Sequence (5'-3') sense/
Antisense gene Size (bp) Annealing temperature
(AT, ℃) One TCCTGATGTTGGTGGCTTCAG S Mmp3
102
58
2 TGTCTTGGCAAATCCGGTGTA AS 3 TGATGGACCTTCTGGTCTTCTGG S Mmp13
473
58
4 CATCCACATGGTTGGGAAGTTCT AS 5 TCACTGCCACCCAGAAGAC S Gapdh 450 58 6 TGTAGGCCATGAGGTCCAC AS

As a result, as shown in Fig. 3A, it was confirmed that the transcriptional expression of Mmp3 and Mmp13 increased by IL-1β in chondrocytes was suppressed in a concentration-dependent manner by the extract of Mt.

3-2. Mmp3 And Mp13 Confirmation of protein expression level inhibition effect

The chondrocytes obtained in Example 2-1 were treated with 1 ng/ml of IL-1β and Mugwort extract at 0, 10, 50 and 100 μg/ml, respectively, for 24 hours to confirm the protein expression levels of Mmp3 and Mmp13. I did. Chondrocytes not treated with anything were used as a control (Con).

The secreted proteins Mmp3 and Mmp13 were reacted with 900 µl of a serum-free conditioned medium with 100 µl trichloroacetic acid (TCA) and then reacted at 0°C for 20 minutes. Then, the supernatant was removed through a centrifuge at 12,000 rpm and 4° C. for 10 minutes, and reacted with 500 μl of cold 100% acetone at 20° C. for 1 hour. 100% acetone and the reacting sample was removed by centrifugation, and the protein was finally precipitated and detected. Western blotting and Western blotting using anti-Mmp3 antibody (Abcam) and anti-Mmp13 antibody (Abcam) The band thickness and concentration were measured using a computer program, and their relative values were expressed with a densitometer.

As a result, as shown in Fig. 3B, it was confirmed that the protein expression of Mmp3 and Mmp13 increased by IL-1β in chondrocytes was decreased in a concentration-dependent manner by the extract of Mugwort. This indicates that the destruction of joints and cartilage tissues can be alleviated or suppressed by the Mugwort Mugwort extract.

Confirmation of the effect of suppressing joint inflammation and cartilage destruction by the extract of P.

Except that the concentration of IL-1β treated on chondrocytes is 5 ng/ml, and the concentrations of the Prunus Ganoderma lucidum extract are 5, 10 and 50 μg/ml, respectively, in the same manner as in Example 3-1. The effect of inhibiting the transcription level of Mmp3 and Mmp13 by mushroom extract was confirmed.

As can be seen in Figure 4, it was confirmed that the transcriptional expression of Mmp3 and Mmp13 increased by IL-1β in chondrocytes was suppressed in a concentration-dependent manner by the extract of P.

Confirmation of suppression of joint inflammation and cartilage destruction by a mixture of Mugwort Mugwort extract and Pleurotus erythraxae extract

Except that the concentration of IL-1β treated in chondrocytes is 1 ng/ml, and 100 μg/ml of Mugwort extract and 0, 10 or 50 μg/ml of P. In the same manner as in Example 3-1, the effect of inhibiting the transcription level of Mmp3 and Mmp13 was confirmed by a mixture of wormwood wormwood extract and Prunus ganoderma lucidum extract.

As a result, as shown in FIG. 5, it was confirmed that the inhibitory effect of the transcription level of Mmp3 and Mmp13 by the mixture of the Mugwort Mugwort extract and the Prunus Ganoderma lucidum extract was significantly superior to the inhibitory effect of each single extract.

Statistical analysis

The results of all examples of the present invention were analyzed using non-parametric statistical methods as data quantified based on an ordinal rating system such as Mankin score. QRT-PCR data expressed as relative fold changes are first checked for the normal distribution using the Shapiro-wilk test, followed by pair-wisecomparisons and multi-comparisons. ), ANOVA (analysis of variance) including Student's t test and post hoc test was used, respectively. Statistical significance was accepted as a probability of 0.05 level (P <0.05).

As described above, specific parts of the present invention have been described in detail, and it will be apparent to those of ordinary skill in the art that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereby. will be. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> REPUBLIC OF KOREA(MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Composition for preventing, improving or treating arthritis comprising extract of Ganoderma lucidum, extract of Artemisia princeps or mixture thereof <130> 1064323 <160> 6 <170> KopatentIn 2.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Mmp3 primer(sense) <400> 1 tcctgatgtt ggtggcttca g 21 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Mmp3 primer (antisense) <400> 2 tgtcttggca aatccggtgt a 21 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Mmp13 primer(sense) <400> 3 tgatggacct tctggtcttc tgg 23 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Mmp13 primer (antisense) <400> 4 catccacatg gttgggaagt tct 23 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Gapdh primer(sense) <400> 5 tcactgccac ccagaagac 19 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Gapdh primer (antisense) <400> 6 tgtaggccat gaggtccac 19

Claims (10)

Ganoderma lucidum ( Ganoderma lucidum ) extract and artemisia princeps ( Artemisia princeps ) extract containing a complex extract mixed in a weight ratio of 1: 2 to 1: 10 as an active ingredient, a pharmaceutical composition for the prevention or treatment of arthritis. The method according to claim 1, wherein the Pyeonggak Gyeongji Mushroom Extract or Mugwort Mugwort Extract is extracted by using water, a lower alcohol of C ₁ to C ₄ or a mixture thereof as a solvent, and the mixture is A pharmaceutical composition for preventing or treating arthritis, characterized in that a mixture. delete The pharmaceutical composition for preventing or treating arthritis according to claim 1, wherein the arthritis is degenerative arthritis or rheumatoid arthritis. The pharmaceutical composition for preventing or treating arthritis according to claim 1, wherein the composition suppresses joint inflammation or cartilage destruction by reducing the mRNA or protein expression of Mmp3 or Mmp13. Ganoderma lucidum ( Ganoderma lucidum ) extract and Artemisia princeps ( Artemisia princeps ) extract containing a complex extract mixed in a weight ratio of 1: 2 to 1: 10 as an active ingredient, a health functional food composition for preventing or improving arthritis. The method of claim 6, wherein the polarization angle Ganoderma lucidum extract or sajabal mugwort extract will extracted by a lower alcohol or a mixture of water, C ₁ to C ₄ as a solvent, the mixture is the polarization angle Ganoderma lucidum extract and sajabal mugwort extract Health functional food composition for preventing or improving arthritis, characterized in that a mixture. delete The health functional food composition for preventing or improving arthritis according to claim 6, wherein the arthritis is degenerative arthritis or rheumatoid arthritis. The health functional food composition for preventing or improving arthritis according to claim 6, wherein the composition suppresses joint inflammation or cartilage destruction by reducing the mRNA or protein expression of Mmp3 or Mmp13.

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