KR101620153B1 - Composition for preventing or treating ostarthritis comprising Glehnia littoralis - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of osteoarthritis or osteoporosis comprising an extract of Aspergillus oryzae as an active ingredient. The present invention relates to a method for preventing or treating osteoarthritis or osteoporosis, which is excellent in reducing or inhibiting the activity of the inflammatory cytokines IL-17, IL-6 or TNF-, In addition, there is no cytotoxicity, there is no toxicity to the drug, and no side effects, so that it can be safely used even when taken for a long time, and it has a stable effect in the body.

Description

[0001] The present invention relates to a composition for preventing or treating arthritis,

The present invention relates to a pharmaceutical composition for the prevention or treatment of osteoarthritis or osteoporosis comprising an extract of Aspergillus oryzae as an active ingredient.

The human body consists of about 200 joints. Joints are the sites where bones and bones meet. The joints are made up of cartilage, joint capsule, synovial membrane, ligament, tendon, and muscle to smoothly move between the bones and the bones, and absorb the impact caused by the movement.

Inflammatory diseases in these joints are caused by chronic rheumatoid arthritis, which is understood to be caused by autoimmunity, infectious arthritis caused by bacterial infection, deformed arthritis caused by degeneration or destruction of articular cartilage or bone due to various causes, And crystalline arthritis in which soluble metabolites are deposited as crystals in the connective tissue around the joints.

Degenerative arthritis, i.e., osteoarthritis, is caused by degeneration of aging and the like in chondrocytes constituting joints, thereby inhibiting the synthesis of type II collagen (type II collagen) and proteoglycan, which are matrix materials of joints in chondrocytes, The production of inflammatory cytokines, such as interleukin-1 and tumor necrosis factor-alpha, has led to the synthesis of matrix metalloproteinases (MMPs) It is a disease caused by destruction of joint tissue due to increase in activity in articular cells.

In addition, arthritis is further exacerbated by the production of nitric oxide by inflammatory cytokines and the production of self-amplifying cytokines by the produced nitric oxide, which leads to the synthesis of more MMPs and accelerates the degradation of joint matrix. At the same time, inflammatory cytokines increase the production of prostaglandin E2, a lipid metabolite, leading to an inflammatory response in arthritis.

Rheumatoid arthritis is a chronic systemic inflammatory disease characterized by symmetrical, multiple arthritis and subsequent joint damage and deformation. In the absence of treatment for rheumatoid arthritis, the course is poor and indicates a disability of the joint function, and if persistent, disability of the joint function interferes with daily life. In Korea, it is estimated that about 1% of the whole population is suffering from rheumatoid arthritis. The incidence of rheumatoid arthritis is three times higher in women than in men, and it is known to occur mainly in the 20s and 40s.

The main causes of rheumatoid arthritis are becoming increasingly evident, and genetic factors, infections, and hormonal abnormalities are thought to be causative factors. Because of these causative factors, 'autoimmune' phenomenon occurs. Autoimmune is a phenomenon that chronic inflammation occurs in many parts of the body due to abnormality of immunity control of our body.

On the other hand, the drugs used in the treatment of arthritis can be classified based on the main mechanism of action such as reduction of inflammation, delay of disease progression, and decrease of uric acid concentration, and many neuroarthritis treating drugs act to reduce inflammation. Inflammation is a pathological process that causes pain, swelling, warmth, seizures, and stiffness. Drugs that rapidly relieve inflammation include steroidal anti-inflammatory drugs, including aspirin, non-steroidal anti-inflammatory drugs and cortisone.

Nonsteroidal anti-inflammatory drugs have the effect of reducing pain and relaxing nerve joints and relieving inflammation. However, because of gastrointestinal disturbances or abdominal pain, it is recommended that non-steroidal anti-inflammatory drugs be given to people with active peptic ulcer or gastrointestinal bleeding Use prohibited. Steroidal anti-inflammatory drugs are not used for degenerative arthritis due to serious side effects such as weight gain and hypertension compared with their effects.

In particular, steroidal anti-inflammatory drugs have nothing to do with the causative treatment of the disease, and may simply induce excessive use of the joints by temporarily reducing the pain, which causes destruction of the nerve joints and deterioration of the disorder. It requires attention.

Therefore, conventional therapies used for joint damage such as arthritis have limited effectiveness, involve obvious toxic side effects, can not be used continuously for a long period of time, and their effectiveness is limited. Therefore, New therapies or therapeutic agents are in desperate need.

Korean Patent No. 656571 Korean Patent No. 585562

Accordingly, the inventors of the present invention confirmed that Glehnia littoralis inhibits inflammatory cytokines and reduces osteoclasts and thus can be used as a therapeutic agent for arthritis or osteoporosis, thus completing the present invention.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating arthritis, which contains Glehnia littoralis as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating osteoporosis, which comprises Glehnia littoralis as an active ingredient.

Another object of the present invention is to provide a health functional food for preventing or ameliorating arthritis or osteoporosis, which comprises Glehnia littoralis as an active ingredient.

It is a further object of the present invention to provide a method for inhibiting osteoclast differentiation, which comprises treating osteoclasts in vitro with Glehnia littoralis extract.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating arthritis, which comprises Glehnia littoralis as an active ingredient.

In one embodiment of the present invention, the Glehnia littoralis extract may be a methanol extract obtained by using methanol.

In one embodiment of the invention, the Glehnia littoralis extract may reduce or inhibit the activity and production of the inflammatory cytokines IL-17, IL-6, interferon-gamma or TNF-a.

In one embodiment of the present invention, the Glehnia littoralis inhibits cartilage destruction by inhibiting expression of MMP1 (metalloproteinase 1), MMP3 (metalloproteinase 3), and MMP13 (metalloproteinase 13) (Tissue inhibitor of matrix metalloproteinase 1) and Timp3 (Tissue inhibitor of matrix metalloproteinase 3), which are cartilage regeneration factors.

In one embodiment of the present invention, the Glehnia littoralis extract may be contained at a concentration of 0.1 ug / ml to 500 ug / ml.

In one embodiment of the present invention, the arthritis may be selected from the group consisting of geriatric arthritis, degenerative arthritis, autoimmune arthritis, osteoarthritis, rheumatoid arthritis, and arthritis associated with obesity or hyperlipemia.

In one embodiment of the present invention, the composition may further comprise coenzyme Q10.

In one embodiment of the present invention, coenzyme Q10 may be contained at a concentration of 3 to 6 uM and cochineal extract may be contained at a concentration of 200 to 300 ug / ml.

In addition, the present invention provides a pharmaceutical composition for preventing or treating osteoporosis, which comprises Glehnia littoralis as an active ingredient.

In one embodiment of the present invention, the composition may further comprise coenzyme Q10.

In addition, the present invention provides a method for inhibiting osteoclast differentiation, comprising treating osteoclasts with Glehnia littoralis extract in vitro.

Further, the present invention provides a health functional food for preventing or ameliorating arthritis or osteoporosis comprising Glehnia littoralis as an active ingredient.

The present invention relates to a composition for preventing or treating osteoarthritis or osteoporosis, which comprises Glehnia littoralis as an active ingredient. The present invention relates to a method for preventing or treating osteoarthritis or osteoporosis, which is excellent in the activity of reducing or inhibiting the activity of the inflammatory cytokines IL-17, IL-6 or TNF-a and osteoclast differentiation, The composition can be used as a composition which can be treated.

In addition, there is no cytotoxicity, toxicity to the drug, and side effects are avoided, so that they can be used safely even when taken for a long period of time and have a stable effect in the body.

FIG. 1 shows the result of obtaining spleen cells from a mouse, treating the extracts of Ganoderma lucidum with concentration, and confirming cytotoxicity.
FIG. 2 is a result of examining the cytotoxicity of PMBC cells isolated from human whole blood by treating the Pseudomonas sp.
FIG. 3 shows the results of measurement of IL-17 and TNF alpha by ELISA after incubation with anti-CD3 0.5 ug / ml or LPS 100 ng / ml on mouse spleen cells and cultured by treatment with various concentrations of Ganoderma lucidum extract.
FIG. 4 shows the results of measuring the amounts of IL-17 and IL-6 by ELISA after culturing PMBC cells isolated from human whole blood with anti-CD3 0.5 g / mL.
FIG. 5 shows the results of confirming the degree of osteoclast differentiation after inducing the differentiation of bone marrow cells of the mouse in the presence of MCSF and soluble RANKL in order to examine arthritis inhibition effect by the extract of Guanxia fusiformes.
FIG. 6 shows the results of real-time PCR observation of osteoclast-related molecules in order to observe the osteoclast inhibitory effect.
FIG. 7 shows the results of measurement of the amounts of IL-17 and IFN-r by ELISA after incubation with anti-CD3 0.5 g / mL or LPS 100 ng / ml in 6-week-old mice and 35-week-old mouse splenocytes.
FIG. 8 shows the effect of IL-17, IFN-r, and TNF-α on the activity of anti-CD 0.5 g / mL or LPS 100 ng / The amount was measured by ELISA.
FIG. 9 shows that PMBC cells isolated from human whole blood cells were cultured at a concentration of 0.5 g / mL of anti-CD3 and then cultured for 48 hours at a concentration of 250 g / mL. The IL-17, IL-6 and IFN- Was measured by ELISA.
FIG. 10 shows the result of measuring the pain of osteoarthritis-induced mice in order to examine the therapeutic effect of osteoarthritis caused by the extracts of Ganoderma lucidum.
FIG. 11 shows the results of Realtime PCR for the expression of molecules related to cartilage destruction catabolic pathway and anabolic pathway-related molecules.
FIG. 12 shows the results of confirming the therapeutic effect and antibody-modulating effect of an animal model of rheumatoid arthritis using a rhizomes.
FIG. 13 shows the effect of the extract of R. japonicus on the treatment of rheumatoid arthritis. The degree of infiltration of inflammatory cells by H & E staining on the joints of rheumatoid arthritis mice (IL-1Rako) .
FIG. 14 shows the results of FACS analysis of the amount of IL-17 expressed in CD-4 cells of spleen cells obtained from a rheumatoid arthritis mouse (IL-1Rako) administered with a 5-week shimadzu.
FIG. 15 shows the results of stimulation of RANKL and M-CSF with TRAP-positive cells in bone marrow cells obtained from a rheumatoid arthritis mouse (IL-1Rako) administered with 5-week shrimp.
FIG. 16 shows results of TRAP-positive cells treated with coenzyme Q10 alone, cochineal extracts, and coenzyme Q10 and cochineal extracts in combination with RANKL and M-CSF to treat bone marrow cells of mice, .

The present invention provides a pharmaceutical composition for preventing or treating arthritis comprising an extract of Glehnia littoralis as an active ingredient.

While studying to develop a new therapeutic agent for treating arthritis, the present inventors have paid attention to the nematocysts, which grow well in the sandy beaches and grow in the sandy beaches. The whole is covered with white hairs and the roots are about 20cm high. The petiole is long and the leaf is a double-leaf type with triangular or oval triangle. Leaves are oval or egg-shaped, thick and shiny, with sawtooth on edge. Flowers are known to bloom in June to July in white.

It is used as a herbal medicine for fever, and it is a special medicine for multiple neuropathic pain and dry bowel syndrome. It is not affected by paralysis every day. It also works when dizziness, headache, tears run down from your eyes, and your limbs are aching and seizures. It is also known that it is effective for colds because it is good for sweating of women, great depression, irregular uterine bleeding and strong sweating.

However, in the past, there has been no mention at all about the fact that the nasal sprays can be used for prevention or treatment of arthritis.

Therefore, the present invention was firstly applied to the present invention, and it was confirmed that the extract of Ganoderma camphorata can be used for the prophylactic or therapeutic treatment of arthritis. In particular, the extract of Ganoderma camphorata inhibits the inflammatory cytokines IL-17, IL-6, interferon gamma or TNF- And osteoclast differentiation was also observed.

More specifically, according to one embodiment of the present invention, MTT analysis of mouse spleen cells and human PBMC cells revealed no cytotoxicity (see FIGS. 1 and 2).

In addition, the present inventors cultured the mouse spleen cells with 0.5 μg / ml of anti-CD3 or 100 ng / ml of LPS through another embodiment, wherein the cells were treated with 25 μg / ml, 100 μg / ml, IL-17 and TNF alpha, which are typical Th17 and Th1 type cytokines, were measured by ELISA in supernatants after culturing at a concentration of 250 ug / ml. As a result, compared with the control group, IL-17 and TNF alpha were significantly decreased (see FIG. 3), and that IL-17 and IL-6 production was also decreased in PBMC cells isolated from human whole blood 4).

Further, the inventors of the present invention have confirmed that the effect of the nematodes on the differentiation of bone marrow macrophages into osteoclasts results in the formation of TRAP-positive multinuclear osteoclasts induced by M-CSF and RANKL, , It was reduced to about half of that of the control group, and it was completely inhibited by the extracts of 100 .mu.g / ml and 500 .mu.g / ml of the bark extract (see FIG. 5).

In addition, TRAP, MMP9, CTR, and NFATc1, which are related to osteoclast, were reduced by the concentration of the extract of R. japonicus by real time PCR, (Fig. 6). As a result of investigating the effect of the extract of Chrysanthemum morifolium extract on the expression of the factors related to the cartilage destruction, it was found that the catabolic-related molecule The expression of MMP1, MMP3, and MMP13 was suppressed by the nematocysts, whereas the expression of TIMP1 and TIMP3, anabolic pathway related molecules related to cartilage regeneration, was increased by the nematodes (see FIG. 11).

In addition, the present inventors isolated spleen cells from a mouse in a young mouse and an old mouse, stimulated the cells with anti-CD3 or LPS to stimulate the production of inflammatory cytokines, And the production of inflammatory cytokines was analyzed. As a result, the production of inflammatory cytokines was significantly inhibited in the group treated with the extracts of P. vivax, compared to the group without treatment.

Therefore, the inventors of the present invention have found that the extract of Ganoderma lucidum can be effectively used for the treatment of geriatric arthritis (see FIGS. 7 and 8).

Furthermore, the inventors of the present invention found that the present invention can prevent or treat osteoporosis induced by osteoclastogenesis. In particular, according to one embodiment of the present invention, bone marrow cells are obtained from a mouse, When treated with RANKL or M-CSF and then administered with the extract of Rhododendron japonicus, the number of TRAP-positive cells, which are an osteoclast factor, was significantly reduced as compared to the group without Rhodobendil extract (see FIG. 15).

Therefore, the inventors of the present invention have found that the present invention can be used for pharmaceutical use for treating arthritis based on the experimental results obtained in the following examples of the present invention.

Specifically, the arthritic condition to which the extract of the present invention can be applied is selected from the group consisting of geriatric arthritis, degenerative arthritis, autoimmune arthritis, osteoarthritis, rheumatoid arthritis and arthritis accompanied by obesity or hyperlipemia .

In addition, the present invention relates to a method for preventing or treating osteoporosis, which comprises administering an effective amount of a compound of formula (I) Can be treated.

As a reference, osteoporosis is one of the bone metabolic diseases. Bone metabolism consists of osteoblasts responsible for osteogenesis and osteoclasts responsible for bone resorption. These cells balance their functions to maintain homeostasis. However, when osteoclast activity is decreased or osteoclast activity is increased, osteoporosis may be induced by decreasing bone density. The causes of osteoporosis are known as women's menopause, hyperthyroidism, diabetes, stress, smoking, lack of exercise, physical aging and taking glucocorticoid-based drugs. In addition, osteoclasts are differentiated by various differentiation factors in macrophage-like precursor cells, and in particular, RANKL (receptor activator of nuclear factor kappa B ligand) secreted from osteoblasts is present on osteoclast precursor cells and osteoclast surface (RANK), which induces the differentiation and activation of osteoclast precursor cells into osteoclasts. The differentiated osteoclasts were treated with TRAP (tartarate resistant acid phosphatase) through activation of NF-B, c-Fos, c-jun, AP-1 and NFATc1 and activation of MAPK, ERK, JNK, p38, Src, cathepsin K, and calcitonin receptor.

Therefore, in order to treat osteoporosis such as osteoporosis, a substance that inhibits osteoclast differentiation or inhibits the expression of osteoclast-specific protein can be used as a therapeutic agent. From this point of view, the present guinea fowl extract can inhibit the differentiation of osteoclasts and reduce the number of TRAP-positive cells, thus being useful as a therapeutic agent for osteoporosis.

In addition, according to the present invention, coenzyme Q10 can be used as a substance that can be used together with a guinea fowl extract for more effective treatment of osteoporosis. According to one embodiment of the present invention, After stimulation of cells with CSF and RANKL, co-treatment of cochineal extracts with coenzyme Q10 showed that the number of TRAP-positive cells was significantly reduced in the co-treated group compared to the treated group.

Also, when coenzyme Q10 was used in combination with the extract of Guanxi guinea pigs, coenzyme Q10 was used at a concentration of 3 ~ 6 uM and guinea fowl extract at a concentration of 200 ~ 300 ug / ml.

In addition, the present invention provides a method for inhibiting osteoclast differentiation, comprising treating bone marrow cells with Glehnia littoralis extract in vitro.

Meanwhile, the Glehnia littoralis according to the present invention can be obtained by extracting and isolating from nature using extraction and isolation methods known in the art, and the extract defined in the present invention can be obtained by using an appropriate solvent For example, a hot-water extract, a polar-solvent-soluble extract or a non-polar-solvent-extractable extract of P. vivax.

As the solvent suitable for extracting the extract from the sea breeze, any solvent acceptable in the art may be used, and water or an organic solvent may be used. Examples of the solvent include alcohols having 1 to 4 carbon atoms, acetone, ether, and the like, including purified water, methanol, ethanol, propanol, isopropanol, butanol, Various solvents such as benzene, chloroform, ethyl acetate, methylene chloride, hexane and cyclohexane may be used alone or in combination. But is not limited to.

As the extraction method, any one of the methods such as hot water extraction method, cold extraction method, reflux cooling extraction method, solvent extraction method, steam distillation method, ultrasonic extraction method, elution method and compression method can be selected and used. In addition, the desired extract may be further subjected to a conventional fractionation process or may be purified using a conventional purification method. There is no limitation on the production method of the present invention, and any known method can be used.

For example, the ginseng extract of the present invention can be prepared in powder form by an additional process such as vacuum distillation, freeze-drying, spray-drying, or the like, with the primary extract extracted by the hot water extraction or solvent extraction method described above have. Further, the primary extract can be further purified by using various chromatographies such as silica gel column chromatography, thin layer chromatography, high performance liquid chromatography and the like, You can get it.

Therefore, the present invention is a concept that includes all the extracts, fractions and tablets obtained in each step of extraction, fractionation or purification, their diluted solutions, concentrates or dried products.

The composition of the present invention comprising such an extract as an active ingredient may be a pharmaceutical composition.

The pharmaceutical composition of the present invention can be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the above-mentioned active ingredients. Examples of the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, A lubricant or a flavoring agent can be used.

The pharmaceutical composition may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the above-described active ingredients for administration.

The pharmaceutical composition may be in the form of granules, powders, tablets, coated tablets, capsules, suppositories, liquids, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation into tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Also, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included as a mixture. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, natural and synthetic gums such as corn sweeteners, acacia, tracker candles or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum and the like. Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile water and sterile water suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

In one embodiment of the present invention, the peanut-shell extract of the present invention may be contained at a concentration of 0.1 ug / ml to 500 ug / ml based on the total weight of the composition.

In addition, the composition of the present invention may also be a food composition. In addition to containing an effective component, the composition of the present invention may contain various flavors or natural carbohydrates such as an ordinary food composition as an additional ingredient have.

Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. The above-described flavors can be advantageously used as natural flavorings (tau martin), stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.).

The food composition of the present invention can be formulated in the same manner as the above 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, chocolates, foods, confectionery, pizza, ram noodles, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes, .

In addition, the food composition may contain various additives such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and flavors such as natural flavors, colorants and aging agents (cheese, chocolate, etc.), pectic acid, Salts of alginic acid and its salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the food composition of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks.

The active ingredient of the present invention is a natural substance, which has little toxicity and side effects. Therefore, it can be safely used for long-term use for the prevention and treatment of arthritis and osteoporosis.

The health functional food of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and circles for the purpose of prevention and treatment of arthritis and osteoporosis.

In the present invention, the term "health functional food" refers to a food prepared and processed by using raw materials or ingredients having useful functions in accordance with Law No. 6727 on Health Functional Foods, and the nutritional control on the structure and function of the human body Or for the purpose of obtaining a beneficial effect for health use such as physiological action.

The health functional foods of the present invention may contain conventional food additives and, unless otherwise specified, whether or not they are suitable as food additives are classified according to the General Rules for Food Additives approved by the Food and Drug Administration, Standards and standards.

Examples of the food items included in the above food additives include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as persimmon extract, licorice extract, crystalline cellulose, high color pigment and guar gum; L-glutamic acid sodium preparations, noodle-added alkalis, preservative preparations, tar coloring preparations and the like.

For example, the health functional food in the form of tablets can be prepared by granulating a mixture obtained by mixing the active ingredients of the present invention with excipients, binders, disintegrants and other additives in a usual manner, Or the mixture can be directly compression molded. In addition, the health functional food of the tablet form may contain a mating agent or the like if necessary.

The hard capsule of the capsule-type health functional food can be prepared by filling a normal hard capsule with a mixture of an effective ingredient of the present invention and a mixture of an additive such as an excipient and an additive such as an excipient. And filling the mixture with a capsule base such as gelatin. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative and the like, if necessary.

The ring-shaped health functional food can be prepared by molding a mixture of the active ingredients of the present invention, a mixture of an extract of Gucci windbreaker, an excipient, a binder and a disintegrant by a known method, and if necessary, Or it may be coated with a material such as starch, talc.

The granular health functional food may be prepared by granulating a mixture of the active ingredients of the present invention with the extract of the present invention, excipient, binder, disintegrant and the like by a conventional method, and if necessary, And the like.

The health functional food may be a beverage, a meat, a chocolate, a food, a confectionery, a pizza, a ramen, a noodle, a gum, a candy, an ice cream, an alcoholic beverage, a vitamin complex and a health supplement food.

Hereinafter, the present invention will be further described by way of examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

Statistical processing

Experimental results were expressed as mean standard deviation. The results were analyzed by variance (ANOVA) using Tokey's test to analyze the differences. A p-value <0.05 was considered statistically significant.

&Lt; Example 1 >

Manufacture of Rhizoctonia sp.

The Glehnia littoralis used in this example was collected directly from Gangneung in September, 2012. After drying in the shade, it was stored at -25 until extraction, and then it was chopped and then cut into 80% methanol (MeOH) After three times of extraction, the mixture was filtered and concentrated under reduced pressure with a rotary evaporator to obtain a methanol-soluble fraction (i.e.

<Experimental Example 1>

MTT analysis

<1-1> Mouse splenocytes

Afforded the spleen (spleen) cells from the mice to investigate whether or arthritis treatment of Conger windproof extract, 10% _{37, 5% CO 2 incubator FBS} , 100 U / mL penicillin, and 100 g / mL streptomycin (streptomycin ). &Lt; / RTI &gt; The mouse spleen cells were treated with anti-dinitrophenyl (DNP) IgE (0.1 g / mL) for 22 hours and treated with anti-dandruff extract at 37 for 15 minutes before the reaction with HSA (1 g / mL). In addition, to observe the cytotoxic ability of the extracts of P. japonicus, it was confirmed by 3- (4,5 dimethyl-thiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) assay according to Mosmann's method. Briefly, the mouse spleen cells were incubated for 24 hours with MTT stock solution (5 mg / mL) and reacted for 4 hours in a 37% 5% CO ₂ incubator. After the reaction, acidic isopropanol was added to 96-well multiplates and read at 560 nm using SpectraCount (Packard Instrument Co. Downers, Inc., Meriden, CT, USA).

As a result, cytotoxicity was not observed even when 500 g / mL of the highest concentration of P. vivax extract was treated (see FIG. 1).

&Lt; 1-2 > Human PBMC cells

Human whole blood (human peripheral blood) separating the PBMC (Peripheral blood mononuclear cell) 10 % at 37, 5% CO ₂ incubator for FBS, 100 U / mL penicillin, and 100 g / mL streptomycin DMEM with neomycin (streptomycin) is included in the Lt; / RTI &gt; The mouse spleen cells were treated with anti-dinitrophenyl (DNP) IgE (0.1 g / mL) for 22 hours and treated with anti-dandruff extract at 37 for 15 minutes before the reaction with HSA (1 g / mL). In addition, to observe the cytotoxic ability of the extracts of P. japonicus, it was confirmed by 3- (4,5 dimethyl-thiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) assay according to Mosmann's method. Briefly, the mouse spleen cells were incubated for 24 hours with MTT stock solution (5 mg / mL) and reacted for 4 hours in a 37% 5% CO ₂ incubator. After the reaction, acidic isopropanol was added to 96-well multiplates and read at 560 nm using SpectraCount (Packard Instrument Co. Downers, Inc., Meriden, CT, USA).

As a result, cytotoxicity was not observed even when 500 g / mL, which is the highest concentration of Ganoderma lucidum extract, was treated (see FIG. 2).

<Experimental Example 2>

The inhibitory activity of IL-17 production by the extract of the present invention

The inventors of the present invention investigated whether the methanol extract of P. aeruginosa inhibits the production of IL-17, an inflammatory cytokine, by the following method.

<2-1> Mouse splenocytes

First, spleen cells were obtained from the mouse, that is, the spleen extracted from the mouse was teased by slicing the spleen tissue using a glass slide, and then erythrocytes in the spleen were removed with a red blood cell hemolysis solution. After that, PBS Splenocytes were obtained by adding a buffer solution and washing by centrifugation.

Then, 1x10 &lt; ⁶ &gt; of single cells isolated from the spleen was cultured with 0.5 [mu] g / ml of anti-CD3 or 100 ng / ml of LPS. At this time, the cells were cultured at a concentration of 25 μg / ml, 100 μg / ml and 250 μg / ml, and then cultured in a supernatant to express typical Th17 and Th1 cytokines IL-17 and TNF alpha Was measured by ELISA.

As a result, the IL-17 and TNF alpha cytokines were significantly decreased in the shark's skin extract-treated group compared with the control group, and the inflammatory cytokine inhibitory activity It was found that the effect was remarkable even when a small capacity was processed (see FIG. 3).

&Lt; 2-2 > Human PBMC cells

Peripheral blood mononuclear cells (PBMC) (1 × 10 ⁶ ) isolated from human peripheral blood were cultured in a 24-well plate coated with 0.5 g / mL of anti-CD3 antibody. At this time, the cells were cultured at a concentration of 25 μg / ml, 100 μg / ml and 250 μg / ml, and the amount of cytokines IL-17 and IL-6 in the supernatant was measured by ELISA Respectively.

As a result, it was shown that IL-17, which is an inflammatory cytokine, was significantly inhibited by the extract of the present invention. In the case of IL-6, which is an inflammatory cytokine, production of IL-6 (See Fig. 4).

<Experimental Example 3>

Analysis of inhibition effect of arthritis by the present invention

The effect of the extracts of P. vivax on osteoclast formation was confirmed by slightly modifying the known method to induce differentiation of mouse bone marrow cells in the presence of MCSF (macrophage colony-stimulating factor) and soluble RANKL (Sugatani et al. 2003, J. Cell. Biochem., 90, 59-67). Bone marrow cells were prepared from the femur and tibia of DBA / 1J mice at 6 weeks of age, and M-CSF (30 ng / ml) was added to the 37-8 chamber slides (3 × 10 ⁵ cells / well; Nalge Nunc International, Naperville, IL) : R & D Systems, Minneapolis, MN). After 3 days, non-adherent cells including lymphocytes were removed and adherent osteoclast precursor cells were further stained with M-CSF (30 ng / ml) and RANKL (30 ng / ml; Strathmann, Hamburg, Germany) Lt; RTI ID = 0.0 &gt; cells / day. &Lt; / RTI &gt; The medium was exchanged once during the incubation with M-CSF and RANKL. Cells were fixed and TRAP (tartrate-resistant acid phosphatase) was stained using a staining kit (sigma) according to the manufacturer's protocol. Microscopic examination of each chamber (40 magnification) showed that TRAP-positive polynuclear cells containing more than 3 nuclei were counted as osteoclasts (Sugatani et al., 2003, J. Cell. Biochem., 90, 59-67).

The cytotoxicity of the extracts of P. japonicus was measured in bone marrow cells and differentiated osteoclasts. Bone marrow cells (1 × 10 ⁵ cells / well) were inoculated into 9 plates and allowed to stand for 24 hours. The cells were allowed to stand for 48 hours after the addition of various concentrations of Ganoderma lucidum extract and the surviving cells were stained with water-soluble tetrazolium salt WST-8 (Cell Counting Kit-8, Donjindo Laboratories, Kumamoto, Japan) according to the manufacturer's protocol. Bone marrow cells were differentiated into osteoclasts and allowed to stand for 48 hours in the presence of M-CSF and RANKL at various concentrations of the extracts of Ganoderma lucidum in order to measure the cytotoxicity of the extracts from the differentiated osteoclasts. Cells were stained for TRAP and counted for TRAP positive polynuclear cells.

Since osteoclasts are known to play a significant role in bone destruction of arthritic joints, the effect of gingival extract on the differentiation of bone marrow macrophages into osteoclasts was confirmed. M-CSF and RANKL-induced TRAP-positive multinuclear osteoclasts were reduced to about half of that of the control group at the concentration of 10 ug / ml, and 100 ug / ml and 500 ug / ml (Fig. 5). The results are shown in Fig.

In addition, TRAP, MMP9, CTR and NFATc1, which are related to osteoclast, were analyzed by real time PCR in RNA obtained from osteoclast inhibition experiments. As a result, ml, respectively (Fig. 6).

<Experimental Example 4>

Inhibitory effect of inflammatory cytokine inhibition by the present invention

The inventors of the present invention investigated whether or not the extract of Rhododendron japonicus extract has an inflammatory cytokine inhibitory effect through the following experiment.

<4-1> Inflammatory cytokine inhibition assay on spleen cells of young and old mice

First, spleen cells were obtained from 6-week-old young mice and 35-week-old old mice. The spleens extracted from the mouse were obtained by finely dividing spleen tissue using a teasing slide, Lt; / RTI &gt;

Then, 1 × 10 ⁶ of single cells isolated from the spleen was cultured for 48 hours with 0.5 g / mL of anti-CD3 or 100 ng / mL of LPS. The cells were incubated with 25 μg / ml, 100 μg / ml and 250 ug / ml, respectively, and the amounts of IFN-r and IL-6, which are representative cytokines IL-17 and Th1 type cytokines, in the supernatant were measured by ELISA.

As a result, IL-17, IFN-r, and IL-6 were significantly reduced in old and young mice compared with the control group without shrimp extract 7). Therefore, it was found that the extract of the present invention of the present invention has an excellent inhibitory effect on inflammatory cytokines not only in young mice but also in old mice, and thus can be effectively used for the treatment of geriatric arthritis.

The present inventors also obtained spleen cells from disease mice (arthritis-inducing mice), and the spleens extracted from the mice were chopped into spleen tissues using a teasing slide, Red blood cells were removed. Splenocytes were then obtained by adding PBS buffer solution and washing by centrifugation.

Then, 1 × 10 ⁶ of single cells isolated from the spleen was cultured with anti-CD3 0.5 g / mL or LPS 100 ng / ml. After culturing the cells in a concentration of 250 μg / ml, IL-17 and Th1-type cytokines IFN-r and TNF alpha were measured by supernatant by ELISA Respectively.

As a result, it was found that the IL-17, IFN-r, and TNF alpha were significantly decreased in the group treated with the shark's pan extract, as compared with the control group (see FIG. 8).

&Lt; 4-2 > Inhibitory effect on inflammatory cytokine production in human PBMC cells

Peripheral blood mononuclear cells (PBMC) (1 × 10 ⁶ ) isolated from human peripheral blood were cultured in a 48-well plate coated with 0.5 g / mL of anti-CD3 antibody. At this time, the cells were cultured for 48 hours at a concentration of 250 ug / ml, and the amounts of cytokines IL-17, IL-6 and IFN-r in the supernatant were measured by ELISA.

As a result, it was shown that IL-17, which is an inflammatory cytokine, was significantly inhibited (about 10 times or more) by the treatment with the extract of the present invention and IL-6 and IFN-r which are inflammatory cytokines (Fig. 9). &Lt; tb &gt;&lt; TABLE &gt;

<Experimental Example 5>

Analysis of the effect of osteoarthritis treatment by the present invention

The inventors of the present invention investigated the degree of improvement of osteoarthritis of Ganoderma lucidum extract by using a degenerative osteoarthritis-inducing model mouse in order to find out whether the extract of Ganoderma sp.

In the degenerative osteoarthritis-induction model mice, 100 mg / kg of Gleevec extract (GLE) was administered. As a positive control, Celecoxib, which is used as a treatment for osteoarthritis, was orally administered at a dose of 5 mg / Was used to dissolve saline. To measure the degree of degenerative osteoarthritis, pain was measured using a dynamic plantar aesthsiometer (Ugo Basile, Comerio, Italy). Machine pain was measured by placing a net plate on the measuring machine, The animals were placed in an animal fixture and then stabbed in the right foot with the drug. After the stabbing, the time (in seconds) it takes the machine to automatically release the foot, and how much weight it takes to release the foot (g).

As a result, it was found that the group to which the present invention of the present invention was injected had the best improvement in osteoarthritis compared to the other groups, and the degree of improvement and treatment was superior to that of Celecoxib, which is conventionally used as a therapeutic agent for osteoarthritis. It was also found that the degree of pain was remarkably reduced to 50% or more within 5 days after the administration of the extract of the present invention (see FIG. 10).

<Experimental Example 6>

Analysis of catabolic and catabolic pathways in THP-1 cells

The present inventors investigated the effect of the present invention on the expression of MMP1, MMP3, and MMP13, which are related to cartilage destruction, and TIMP1 and TIMP3, which are anabolic pathway related molecules, by Realtime PCR.

In vitro, human-derived chondrocyte was pretreated for 24 hours to stabilize the cells. IL-1b was stimulated to stimulate the activity of the cells. The cells were cultured for 24 hours, and RNA was obtained from the cells. Expression of MMP1, MMP3, MMP13, TIMP1 and TIMP3 was observed by real time PCR. At this time, the control group was a group not treated with the bark extract.

As a result, the expression of MMP1, MMP3, and MMP13 was markedly decreased in the group treated with shark's skin extract, whereas the expression of Timp1 and Timp3 associated with cartilage remodeling was significantly increased (see FIG. 11) ).

<Experimental Example 7>

Effect of the present invention on the treatment of rheumatoid arthritis

The present inventors used a 12-week-old IL-1Rako mouse, which has a rheumatoid arthritis index of 6, which is spontaneously arthritis caused by lack of IL-Ra in order to confirm the therapeutic effect of rheumatoid arthritis. The effects of oral ingestion on arthritis were investigated by the following methods.

<7-1> Effectiveness of treatment of autoimmune arthritis by shade wind

The animal model of rheumatoid arthritis prepared above was administered with a rhizome and the symptoms of autoimmune arthritis were observed for 5 weeks.

Since the arthritis model is naturally occurring arthritis rather than a general arthritis-inducing model, it can be seen that the treatment activity of the arthritis gangs is great (see FIG. 12).

<7-2> Analysis of Humoral Response by Humoral Wind

In order to observe the humoral respone in rheumatoid arthritis animal model, the mice were sacrificed at 5 weeks after the administration, and the amount of total IgG and IgG2a expressed in the serum was analyzed by ELISA. Both total IgG and IgG2a involved in the Th1 response were found to be reduced in all of the group of rhizomes (FIG. 12).

<7-3> Effectiveness of treatment of rheumatoid arthritis

In order to observe the treatment effect of the rheumatoid arthritis animal model using the rhizome of the rhizome, the mice were sacrificed after 12 weeks of IL-1Rako mouse administration for 5 weeks and the joints were obtained. The infiltration of the inflammatory cells by H & E staining Respectively.

As a result, it was confirmed that the inflammatory cells were decreased in the group treated with the nasal spray (see Fig. 13).

In addition, the degree of destruction of cartilage was observed with Safranin O. As a result, it was confirmed that cartilage was retained in the brachyury group, but the cartilage was destroyed in the joints of the control group, so that it was not stained.

<7-4> Confirmation of the inhibitory effect of Th17 cells on IL-1Rako mouse splenocyte induced by shading

In order to confirm the therapeutic effect of the rheumatoid arthritis animal model using the rhizome windbreak, 12-week-old IL-1Rako mice were administered with 5 days rhythm of windshield and mice were sacrificed to obtain splenocytes. The expression of IL-17 in CD4 cells of the spleen cells obtained was examined by FACS. As a result, it was observed that the expression of Th17 cells, which is a pathologic cell, was significantly decreased in the mice treated with the Fusarium spp. (FIG. 14).

<7-5> Observation of osteoclast differentiation regulation ability

In order to confirm the therapeutic effect of the animal model of rheumatoid arthritis using the rhizome, the 12-week-old IL-1Rako mouse was administered to the mice for 5 weeks, and the mouse bone marrow cells were obtained. RANKL and M-CSF were stimulated in bone marrow cells of each group, and TRAP-positive cells were examined. As a result, it was observed that TRAP-positive cells were decreased in the group of rhizomes (FIG. 15).

<Experimental Example 8>

Inhibition of osteoclast differentiation by the combination of coleoptera extract and coenzyme Q10

Furthermore, the present inventors searched for a substance that can be compounded to further improve osteoclast differentiation inhibition effect of the present invention. In addition, the inventors of the present invention found that osteoblast extract and coenzyme Q10, And the degree of the number of labeled cells was measured.

For this purpose, M-CSF and RANKL were treated with bone marrow cells used in Experimental Example 7 to stimulate the cells, and coenzyme Q10 alone was treated (5 uM) for each cell, (250 ug / ml), and coenzyme Q10 (5 uM) and bark extract (250 ug / ml) were used in the other groups. Then, the number of TRAP positive cells was measured.

As a result, as shown in Fig. 16, in the group treated alone with coenzyme Q10 (5 uM) and bark extract (250 ug / ml), the number of positive labeled cells of TRAP decreased compared to the group treated with no coenzyme Q10 However, in the case of the combination treatment group, there was almost no positive number of TRAP-labeled cells.

Therefore, the inventors of the present invention have found that when the extract of the present invention is used together with coenzyme Q10, it can effectively inhibit osteoclast differentiation and effectively treat diseases such as osteoporosis, which is a bone disease caused by osteoclast differentiation And it was found.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (13)

A pharmaceutical composition for preventing or treating arthritis, which comprises methanol extract of Ganoderma lucidum (Glehnia littoralis) and coenzyme Q10 as an active ingredient,
The pharmaceutical composition for the prevention or treatment of arthritis according to claim 1, wherein the extract comprises a concentration of 200 ~ 300 ug / ml of coenzyme Q10 and 3 ~ 6 uM of coenzyme Q10. delete delete The method according to claim 1,
The methanol extract of Ganoderma lucidum suppresses cartilage destruction by inhibiting the expression of MMP1 (metalloproteinase 1), MMP3 (metalloproteinase 3), and MMP13 (metalloproteinase 13), which are cartilage destruction factors, of matrix metalloproteinase 1) and Timp3 (Tissue inhibitor of matrix metalloproteinase 3) to promote cartilage regeneration activity. delete delete delete delete A pharmaceutical composition for preventing or treating osteoporosis comprising methanol extract of Ganoderma lucidum (Glehnia littoralis) and coenzyme Q10 as an active ingredient,
The pharmaceutical composition for preventing or treating osteoporosis according to claim 1, wherein the extract comprises a concentration of 200 ~ 300 ug / ml of coenzyme Q10 and 3 ~ 6 uM of coenzyme Q10. delete delete A method for inhibiting differentiation of osteoclasts, comprising the step of treating a bone marrow cell with an extract of Ganoderma lucidum methanol extract (Glehnia littoralis) and Coenzyme Q10 in vitro. A health functional food for preventing or ameliorating arthritis or osteoporosis comprising methanol extract of Ganoderma lucidum (Glehnia littoralis) and coenzyme Q10 as an active ingredient.

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