KR101577792B1 - A pharmaceutical composition for preventing or treating IL-6-mediated disease, comprising extract or fraction of Portulaca oleracea L. - Google Patents

Abstract

The invention Portulaca Oleracea (Portulaca 6. A pharmaceutical composition for the prevention or treatment of an IL-6 (interleukin-6) mediated disease, comprising an extract of Oleracea L. or a fraction thereof as an active ingredient, and a step of administering the composition to a suspect individual with IL-6 mediated diseases Gt; IL-6 < / RTI > mediated diseases. The present invention also relates to a food composition and a quasi-drug composition for preventing or ameliorating an IL-6 mediated disease, comprising an aspartame extract or a fraction thereof as an active ingredient.

Description

TECHNICAL FIELD [0001] The present invention relates to a pharmaceutical composition for preventing or treating an IL-6 mediated disease comprising an extract of Aspergillus oryzae or a fraction thereof as an active ingredient.

The invention Portulaca Oleracea (Portulaca 6. A pharmaceutical composition for the prevention or treatment of an IL-6 (interleukin-6) mediated disease, comprising an extract of Oleracea L. or a fraction thereof as an active ingredient, and a step of administering the composition to a suspect individual with IL-6 mediated diseases Gt; IL-6 < / RTI > mediated diseases. The present invention also relates to a food composition, a quasi-drug composition, a feed additive, or a drinking water additive for preventing or ameliorating an IL-6 mediated disease comprising an aspartame extract or a fraction thereof as an active ingredient.

Interleukin-6 (IL-6), a cytokine, also called B-cell stimulating factor 2 (BSF2) or interferon-2 (IFN-2), was found to be a differentiator involved in the activation of B lymphocytes. It has since been found to be a multifunctional cytokine that affects various cell functions. IL-6 carries its biological activity on the cell membrane through two types of proteins. One is the IL-6 receptor, a protein that IL-6 binds. The IL-6 receptor is expressed through the membrane and is a membrane-bound protein with a molecular weight of about 80 kDa. The other is the membrane protein gp130, which has a molecular weight of about 130 kDa, which belongs to signaling of non-ligand binding. IL-6 and IL-6 receptors form an IL-6 / IL-6 receptor complex, which in turn binds to gp130. After binding of these ligands and receptors, JAK2 (Janus Kinase 2) is activated in the cell by transphosphorylation. Activated JAK2 phosphorylates several tyrosine residues of the cytoplasmic domains and is responsible for signal transducers and activators of STAT3, which have SH2 or other phosphotyrosine binding motifs transcription 3) as a docking site for proteins in the cytoplasm. STAT3 bound to the cytoplasmic domain of the receptor is phosphorylated by JAK2 and then released from the receptor. These activated STAT3 bind to each other in the cytoplasm to form a homodimer or a heterodimer, Into the recognition sequence of the target gene to increase transcription.

Various studies have been conducted on the relationship between IL-6-induced signal transduction and inflammatory diseases, autoimmune diseases and osteoporosis. Therefore, studies for inhibiting the signal transduction system induced by IL-6 for the treatment of inflammatory, autoimmune diseases and osteoporosis are being actively carried out. At present, the inhibition of the signal transduction system of IL- R-antibody (anti-IL-6 receptor antibody). (WO 96/011020) for the treatment of rheumatoid arthritis with said anti-IL-6 R antibody (WO 96/011020), transglutathione, hyperimmunoglobulinemia, anemia, nephritis, Treatment of diseases caused by IL-6 products such as cachexia, rheumatoid arthritis, cattle disease and angina pectoris is already known (WO 96/012503). In addition, the anti-IL-6 R antibody is useful for the treatment of sensitive T-cell related diseases such as multiple sclerosis, uveitis, chronic thyroiditis, delayed hypersensitivity and atopic dermatitis (WO 98/042377), systemic erythromycin, Treatment of Crohn's disease (International Patent Publication No. 99/047170), treatment of pancreatitis (WO 00/010607), treatment of psoriasis (WO 02/034292) and inflammatory chronic arthritis (International Patent Publication No. 02/080969). However, when the anti-IL-6R antibody is introduced into an individual, it may have an epitope which is a part that can be recognized as an exogenous protein. Therefore, it has a disadvantage that it still acts as an immunogen when used as a therapeutic agent. In order to solve the above problems, many studies have been conducted to develop a therapeutic agent for IL-6-mediated diseases using a small molecule compound that is not a protein not recognized by the immune system.

It is known that STAT3 (signal transducers and activators of transcription 3) plays an important role in NF-κB as another important transcription factor in autoimmune diseases including osteoarthritis or rheumatoid arthritis. In particular, STAT3 is known to be activated by the cytokine IL-6 and is also known to be activated by epidermal growth factor (EGF). Recently, studies on the relation between osteoporosis and IL-6 have been reported, and it has been proved through animal models that STAT3 activity induced by IL-6 plays an important role in osteoclast differentiation and bone formation (Bone 2006, 39, 505-512). In STAT3-deficient mice, bone mineral density and bone volume were decreased, and osteoclast numbers were observed to have a negative effect on osteoporosis (BBRC 2005 , 328, 800-807). On the other hand, when madindoline A, a compound that inhibits IL-6-induced STAT3 phosphorylation, was treated with OVX mouse, an animal model for postmenopausal osteoporosis, activity was shown to inhibit bone resorption In the IL-6 - / - mouse model, there was no change in bone loss and osteoclast precursor counts due to estrogen depletion (EMBO J 1994, 13, 1189-1196). This fact indicates that IL-6 plays an important role in bone formation and reabsorption, in particular through phosphorylation of STAT3.

Accordingly, the present inventors have made intensive efforts to find a natural product having a therapeutic effect on IL-6 mediated diseases by targeting the STAT3 pathway activated by IL-6. As a result, the extracts and their fractions inhibited the transcriptional activity and phosphorylation of STAT3 To effectively inhibit the signal transduction system mediated by IL-6, and in particular to have an activity of effectively inhibiting osteoclast differentiation and bone resorption, thereby completing the present invention.

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating an IL-6 mediated disease comprising an aspartame extract or a fraction thereof as an active ingredient.

Another object of the present invention is to provide a method for the treatment of IL-6 mediated diseases, comprising the step of administering the pharmaceutical composition to a suspect individual with an IL-6 mediated disease.

It is still another object of the present invention to provide a food composition for preventing or ameliorating an IL-6 mediated disease, comprising an aspartame extract or a fraction thereof as an active ingredient.

It is still another object of the present invention to provide a quasi-drug composition for preventing or ameliorating an IL-6 mediated disease, which comprises an aspartame extract or a fraction thereof as an active ingredient.

Another object of the present invention is to provide a feed additive or a drinking water additive for preventing or ameliorating an IL-6 mediated disease, comprising an aspartame extract or a fraction thereof as an active ingredient.

The present invention as an embodiment for achieving the above object is Portulaca Oleracea (Portulaca (interleukin-6) mediated diseases, which comprises an extract of oleracea L. or a fraction thereof as an active ingredient.

As used herein, the term "Portulaca Oleracea (Portulaca oleracea L.) "is an annual plant belonging to the genus Pyralidae of the phytoplankton, and is also referred to as Pseudomonas aeruginosa, Although it is known to be effective, the horseradish extract or its fractions have not been known for therapeutic use in IL-6 mediated diseases, particularly inflammatory diseases, autoimmune diseases and bone metabolic diseases, and were first identified by the present inventors. In the present invention, marinated horses can be commercially sold, purchased from nature or cultivated.

In the present invention, the term " marchy extract "means an extract obtained by extracting marchy. The mashin extract has a volume of water ranging from about 2 to 20 times, preferably about 3 to 5 times the dry weight of the gristle-like crushed product, a mixture of 1 (C ₁ ) to 6 (C ₆ ) carbon atoms such as methanol, ethanol, Of a polar solvent of an alcohol of 1 to 4 carbon atoms, more preferably a polar solvent of a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof having a mixing ratio of about 1: 0.1 to 1:10, is used as an elution solvent, Extraction can be carried out using an extraction method such as hot water extraction, cold extraction, reflux cooling extraction or ultrasonic extraction for about 12 hours to 4 days, preferably 3 days, at 100 ° C, preferably at room temperature, -6 < / RTI > mediated diseases. Preferably, the extract is continuously extracted 1 to 5 times by cold extraction, filtered under reduced pressure, and the filtrate is concentrated under reduced pressure at 20 to 100 캜, more preferably at room temperature, in a vacuum rotary condenser, But the present invention is not limited thereto. The diluent, concentrate or extract of the extract, the extract, and the extract may be dried , Or a controlled preparation or a purified product thereof. The mashin extract can be extracted from various organs of natural, hybrid, and variant plants and extracted from plant tissue cultures as well as root, top, stem, leaf, flower, fruit body, . In one embodiment of the present invention, ethanol was added to the powdered marigold, and the mixture was frost-extracted for 7 days (Example 1).

The term "fraction" in the present invention means a product obtained by a fractionation method for separating a specific component or a specific group from a mixture containing various constituents. The marigold fraction of the present invention can be obtained by suspending the marigold extract with water and then fractionating it with a nonpolar solvent such as hexane or ethyl acetate to obtain a polar solvent fraction and a nonpolar solvent fraction, respectively. Specifically, the crude horseradish extract is suspended in distilled water, and a nonpolar solvent such as hexane and ethyl acetate at about 1 to 100 times, preferably about 1 to 5 times the volume of the suspension is added to the suspension for 1 to 10 times, The non-polar solvent soluble layer can be extracted and separated two to five times. In addition, conventional fractionation processes can also be performed (Harborne JB Phytochemical methods: A guide to modern techniques of plant analysis, 3rd Ed. P6-7, 1998). More specifically, after suspending the mashimono extract in water, each solvent-soluble extract can be obtained by continuous extraction using an equal amount of n -hexane and ethyl acetate, and more specifically, a suspension and then, the same amount of n - the fraction was mixed with hexane to n - hexane-soluble fraction and the number and availability fraction can be obtained, the in-soluble fraction was added to ethyl acetate to give the ethyl acetate-soluble fraction and soluble fraction can do. In one embodiment of the present invention, the horseradish extract was suspended in water to obtain water fractions, hexane fractions and ethyl acetate fractions (Example 1-2).

The mashin extract of the present invention and its fractions exhibit STAT3 (signal transducers and activators of transcription 3) inhibitory activity and can be effectively used for the prevention or treatment of IL-6 mediated diseases.

The term "IL-6 mediated disease" in the present invention means a disease that is activated by a signal transduction pathway caused by IL-6, and specifically refers to diseases caused by activation of STAT3 phosphorylation by IL-6 It means disease. For the purpose of the present invention, the IL-6 mediated diseases include, but are not limited to, those diseases or disorders that can be prevented or treated by the mycorrhizal extract of the present invention or fractions thereof, such as inflammatory diseases, autoimmune diseases, But is not limited thereto. Specifically, IL-6 binds to receptors such as gp130 and gp80, resulting in phosphorylation of STAT3 protein, whereby the activated STAT3 protein enters the nucleus and expresses the target gene, thereby causing autoimmune disease, inflammatory disease or bone disease Leading to the onset or progression of metabolic diseases.

The term "inflammatory disease" in the present invention collectively refers to diseases with inflammation as a main lesion, and may mean inflammatory diseases mediated by the IL-6-activated STAT3 for the purpose of the present invention. Examples of the inflammatory diseases include inflammatory bowel disease, transglottial hyperplasia, hyperimmunoglobulinemia, nephritis, cachexia, cattle disease, vasectomy nephritis, multiple sclerosis, uveitis, chronic thyroiditis, delayed hypersensitivity, allergy, contact dermatitis, , Inflammatory idiopathic atrophy, or Alzheimer's. Since the STAT3 protein activated in inflammatory diseases acts as a major transcription factor and leads to the development and progression of inflammatory diseases, the composition of the present invention which effectively inhibits activation of STAT3 protein is useful for prevention and treatment of inflammatory diseases have.

In the present invention, the term "autoimmune disease" means a disease in which an immune response to a self-antigen of a pathological individual is directly or indirectly caused by the STAT3 for activation of IL-6 Mediated autoimmune disease. Such autoimmune diseases may include, but are not limited to, autoimmune pancreatitis, asthma, atopic dermatitis, systemic erythromatosis or psoriasis.

The term "bone metabolic disease" in the present invention means a bone related disease caused by imbalance of osteoblasts and osteoclasts. Examples of such diseases include osteoporosis caused by excessive osteoclast bone resorption, arthritis, periodontal disease, pathological bone diseases such as fracture or Paget disease, However, the present invention is not limited to the above-mentioned bone metabolic diseases.

Among them, "osteoporosis" means a condition in which the amount of bone is reduced and the quality of bone is weakened due to qualitative change, thereby causing a high possibility of fracture. The bone functions as a reservoir for protecting various organs in the body and for storing the necessary substances in the body such as calcium, and a balance between osteoclast that breaks down bone existing in bone tissue and osteoblast that produces bone Followed by homeostasis. In the case of osteoporosis, the balance of the two cell activities is broken, and bone destruction by osteoclast occurs and it develops and progresses. The role of RANKL in the regulation of osteoclast differentiation and activation of various transcription factors, especially IL-6, is shown in RANKL (Kim K et al., J Immunol 2007 May 1; 178 (9): 5588-94). Therefore, a substance capable of inhibiting signal transduction activated by IL-6 and inhibiting osteoclast differentiation and reducing osteoclast-induced bone destruction can be used as a therapeutic agent for osteoporosis. The crude extract of the present invention or its fractions effectively inhibits RANKL-induced inhibition of osteoclast differentiation, effectively inhibits bone resorption and effectively restores bone density and the like, and thus can be effectively used for prevention or treatment of osteoporosis.

The term "arthritis" refers to joint disease that involves inflammation at one or more joint sites. The common form of arthritis is osteoarthritis, which causes inflammation and pain due to damage to the bones and ligaments of the joint due to gradual damage or degenerative changes of the cartilage that is reporting the joint. For the purpose of the present invention, the arthritis is a disease accompanied by loss of bones in the joint region, which may be, for example, osteoarthritis or rheumatoid arthritis.

The term "periodontal disease" refers to inflammatory conditions of the tooth supporting tissues caused by bacteria, which can be separated into gingivitis and periodontitis. The cause of the disease is that oral bacteria caused by poor oral hygiene form to form a bacterial membrane. Tooth surface bacteria is a bacterial mass that grows after the bacteria stick to the surface of the teeth by using a sticky substance in the needle as an adhesive. If you do not leave the bacterial membrane in the tooth, inflammation occurs, sometimes gum bleeding, bad breath, and sometimes the symptoms of gingivitis is called. As the gingivitis further progresses, the gap between the tooth and the gum becomes deeper, resulting in a periodontal pouch, where bacteria that cause periodontal disease multiply and periodontitis occurs. As periodontitis progresses, mild irritation, such as brushing, may cause bleeding from the gums and swelling, often causing acute inflammation and pain. Such inflammation lowers the function of making bone and increases the action of absorbing bone, so that the alveolar bone is gradually lowered, causing the alveolar bone to be destroyed and eventually to lose teeth.

The "fracture" refers to fracture of the bone in a state where the continuity of the bone, alveolar plate, or joint surface is abnormally broken. Causes of fractures include trauma such as traffic accidents, safety accidents caused by industrial disorders, changes in bones due to diseases such as osteoporosis, bone cancer, metabolic diseases, and repeated bones due to sports or loads. In addition, fracture status can be classified as fracture, greenstick fracture, transverse fracture, involuntary fracture, spiral fracture, fracture fracture, fractured fracture, and avulsion fracture based on fracture line (line along the bone tip generated by osteotomy) , Compression fracture, and hamstring fracture.

The "Paget's disease" refers to a localized bone disease in which bone formation is excessively aggravated and the skeletal system of a wide area is invaded. The pathologic mechanism of Paget's disease is believed to be a combination of an excessive increase in bone resorption by osteoclasts with the ability to cleanse the bone and an increase in new bone formation by osteoblasts with bone-forming functions In addition, newly formed bones in the bone jets are structurally disordered and are known to be vulnerable to bone deformation and fracture.

In the prevention or treatment of the above-mentioned bone metabolic diseases, the extracts of the present invention or fractions thereof inhibit the differentiation of osteoclasts and exhibit an activity of inhibiting bone resorption of osteoclasts, Effect can be obtained.

The term "prophylactic" as used herein refers to all actions that inhibit or delay the onset of IL-6 mediated disease by administration of the composition, and "treatment" It means any act that improves or changes the symptoms.

The pharmaceutical composition comprising the crude extract of the present invention or a fraction thereof may further comprise an appropriate carrier, adduct or diluent conventionally used in the production of a pharmaceutical composition. At this time, the content of the gingival extract or its fraction contained in the composition is not particularly limited, but it may be 0.0001 to 10% by weight, preferably 0.001 to 1% by weight based on the total weight of the composition .

The pharmaceutical composition may be in the form of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories May have one formulation, and may be various forms of oral or parenteral administration. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may contain one or more excipients such as starch, calcium carbonate, sucrose or lactose lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The composition of the present invention may be administered in a pharmaceutically effective amount.

The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will vary depending on the species and severity, age, sex, The type of drug, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And can be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art. The preferred dose of the composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration, and the period of time. However, for the desired effect, the extract of the present invention or fraction thereof is administered at a dose of 0.0001 to 100 mg / kg, preferably 0.001 to 100 mg / kg. The administration may be carried out once a day or divided into several times. The composition may be administered to various mammals such as rats, livestock, humans, and the like in a variety of routes. The mode of administration is not limited as long as it is a conventional method in the art. For example, oral, rectal or intravenous, Subcutaneous, intra-uterine dural or intracerebral injection.

In addition, the pharmaceutical composition may be applied to any animal that requires prevention or treatment of an IL-6 mediated disease regardless of its type, and may be applied as an animal medicine for animals.

In one embodiment of the present invention, the chromatographic fractionation of the extracts or fractions of representative marigolds, the ethanol extract of march, its hexane, ethyl acetate fraction and hexane fraction effectively inhibits IL-6-induced STAT3 activity 1 to 3), indicating no cytotoxicity (Fig. 4). In addition, it was confirmed that the horseradish ethanol extract, hexane fraction and ethyl acetate fraction of the present invention effectively inhibited osteoclast differentiation induced by RANKL (FIGS. 5 and 6). In addition, the mechanism of inhibition of osteoclast differentiation by MCH-ethanol extracts showed that MCH-ethanol extract inhibited mRNA expression of c-Fos and NFATc1, which are transcription factors induced by RANKL, in a concentration-dependent manner, MRNA expression of TRAP and OSCAR, which are important genes in the differentiation of Pseudomonas aeruginosa, was effectively inhibited by the ethanol extract of mashin (Fig. 7). In addition, it has been known that MAPK (p38, JNK, ERK), AKT, and IκB are phosphorylated as a signal transducer of RANKL in osteoclast differentiation. As a result, RANKL-induced AKT and IκB phosphorylation but did not affect the phosphorylation activity of MAPK (p38, JNK, ERK) (FIG. 8). In addition, the ethanol extract of mashihyon inhibited the osteoclast differentiation as well as osteoclast differentiation at a concentration of 50 占 퐂 / ml (Fig. 9). Also, in the animal model of LPS-induced bone resorption mouse, , And showed excellent effects of improving both the amount of the postural bone, the area of the posterior space, and the number of the posterior marrow (FIG. 10). From these results, it can be seen that the mashin extract of the present invention and its fractions can be effectively used for the prevention or treatment of diseases mediated by IL-6, such as inflammatory diseases, autoimmune diseases or bone metabolic diseases, Suggesting that the present invention is applicable to the treatment of various bone metabolic diseases caused by excessive action of osteoclasts due to bone loss.

In another aspect, the invention provides a method of treating IL-6 mediated diseases, comprising the step of administering the composition to a suspected individual with an IL-6 mediated condition.

The above compositions, IL-6 mediated diseases and treatments are as described above.

Specifically, the method of treatment of the present invention comprises administering the pharmaceutical composition in a pharmaceutically effective amount to a subject suspected of having an IL-6 mediated condition. The term refers to whole mammals including dogs, cows, horses, rabbits, mice, rats, chickens or humans, but the mammal of the present invention is not limited by these examples. The pharmaceutical compositions may be administered by non-oral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal routes and may be administered by a suitable method, including localized administration, if necessary, for localized treatment. The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and body weight of the individual, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art.

In another aspect, the present invention provides a food composition for preventing or ameliorating an IL-6 mediated disease comprising an aspartame extract or a fraction thereof as an active ingredient.

The marigolds, extracts, fractions and IL-6 mediated diseases are as described above.

Specifically, the extract or fraction of the present invention may be added to a food composition for the purpose of preventing or ameliorating STAT3-mediated diseases, preferably for the prevention or amelioration of autoimmune diseases, inflammatory diseases or osteoporosis.

When the extract is used as a food additive, the extract or the fraction thereof may be used as it is or may be used together with other foods or ingredients, and may be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be appropriately determined depending on the purpose of use.

The kind of the food of the present invention is not particularly limited. Examples of the food to which the extract or its fractions can be added include meat products, sausages, breads, chocolates, candies, snacks, confectionery, pizza, ramen noodles, dairy products including gums, ice cream, Tea, a drink, an alcoholic beverage, and a vitamin complex, and can include foods in a conventional sense, and foods used as feed for animals.

In addition to the above, the food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, , A carbonating agent used in a carbonated drink, and the like. It may also contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. The food may also be prepared in the form of tablets, granules, powders, capsules, solutions in liquid form, and the like according to known production methods. There are no particular restrictions on other ingredients other than those containing the mashin extract or the fraction thereof according to the present invention as an active ingredient, and may include various conventional flavors or natural carbohydrates as an additional ingredient.

In another aspect, the present invention provides a quasi-drug composition for preventing or ameliorating an IL-6 mediated disease, comprising an aspartame extract or a fraction thereof as an active ingredient.

The above-mentioned marigold, its extracts, fractions and IL-6 mediated diseases are as described above.

Specifically, the crude extract of the present invention or its fractions may be added to the quasi-drug composition for the purpose of preventing or improving IL-6 mediated diseases.

The term "quasi-drug" in the present invention means a fiber, a rubber product or the like used for the purpose of treating, alleviating, treating or preventing a disease of a human or an animal, a weak action on the human body, Or products similar to those which are not machinery, preparations used for sterilization, insecticides and similar uses for the prevention of infections, for the purpose of diagnosis, treatment, alleviation, treatment or prevention of diseases of human beings or animals Machinery, or apparatus, and that is not an apparatus, machine, or apparatus of an article used for the purpose of giving pharmacological effects to the structure or function of a person or animal, It also includes supplies.

When the crude extract of the present invention or a fraction thereof is added to a quasi-drug composition for the purpose of preventing or ameliorating an IL-6 mediated disease, the extract or fraction may be directly added or used in combination with other quasi-drugs, It can be used appropriately. The amount of the active ingredient to be mixed can be appropriately determined depending on the purpose of use.

The external preparation for skin is not particularly limited, but may be preferably used in the form of an ointment, a lotion, a spray, a patch, a cream, a powder, a suspension, a gel or a gel. Such personal care products include but are not limited to soap, cosmetics, wet tissue, tissue paper, shampoo, skin cream, face cream, toothpaste, lipstick, perfume, makeup, foundation, ball touch, mascara, eye shadow, sunscreen Lotions, hair care products, air freshner gels or cleaning gels. Further, another example of the quasi-drug composition of the present invention is a disinfectant cleaner, a shower foam, a gagrin, a wet tissue, a detergent soap, a hand wash, a humidifier filler, a mask, an ointment agent or a filter filler.

In another aspect, the present invention provides a feed additive or a drinking water additive for preventing or ameliorating an IL-6 mediated disease, comprising an aspartame extract or a fraction thereof.

The above-mentioned marigold, its extracts, fractions and IL-6 mediated diseases are as described above.

The term "feed additive " in the present invention is a generic term for substances added to animal feeds for nutritional or specific purposes in small quantities. In the present invention, for the purpose of preventing or improving IL-6 mediated diseases Means a substance to be added. Here, the term " animal " is a concept including livestock and pets.

The feed additive of the present invention may further contain a binder, an emulsifying agent, a preservative, and the like added to prevent quality deterioration. The feed additive of the present invention may contain amino acid, vitamin, enzyme, probiotics, A compound, a silicate, a buffer, a coloring agent, an extracting agent, an oligosaccharide, and the like, and may further include, but is not limited to, a feed mixture.

The term "additive for drinking water" in the present invention is a generic term for substances added to drinking water intended for animals or for specific purposes for a specific purpose. In the present invention, for the purpose of preventing or improving IL-6 mediated diseases Means a substance to be added. Here, the term " animal " is a concept including livestock and pets.

The extracts of the present invention or fractions thereof effectively inhibit the signal transduction by IL-6 and effectively inhibit the differentiation of osteoclasts without any adverse effects derived from natural products that have been used for a long time as natural medicines. May be usefully used in the prevention or treatment of IL-6 mediated diseases such as inflammatory diseases, autoimmune diseases or osteoporosis.

FIG. 1 is a graph showing the inhibitory activity of IL-6-induced luciferase in the mashin ethanol extract, hexane and ethyl acetate fractions.
2 is a graph showing the inhibitory activity of IL-6-induced luciferase in the primary column chromatographic fraction obtained from the marine ethyl acetate fraction.
FIG. 3 is a graph showing the results of western blotting of IL-6-induced STAT3 phosphorylation inhibitory activity of horseradish ethanol extracts.
FIG. 4 is a graph showing the results of confirming the toxicity of the mashin ethanol extract, hexane and ethyl acetate fractions.
FIG. 5 is a graph showing the RANKL-induced osteoclast differentiation inhibitory activity of the mashin ethanol extract.
FIG. 6 is a graph showing RANKL-induced osteoclast differentiation inhibitory activity of marine hexane, ethyl acetate and water fractions.
FIG. 7 is a graph showing that the marginal ethanol extract of the present invention inhibits mRNA expression of genes necessary for differentiation of RANKL-induced osteoclasts.
FIG. 8 is a graph showing that the ethanolic extract of Ganoderma lucidum suppresses the differentiation of osteoclasts by inhibiting the phosphorylation mechanism of AKT and IκB, which are lower signal transduction mechanisms of RANKL.
FIG. 9 is a graph showing the activity of hatching ethanol extract inhibiting osteoclast bone resorption.
Fig. 10 shows the effect of gouty ethanol extracts on the treatment of osteoporosis in an animal model of LPS-induced bone resorption.

Hereinafter, the present invention will be described in more detail in the following Examples and Experimental Examples. However, these examples are provided only to aid understanding of the present invention, and the present invention is not limited thereto.

Example : Machi  Extract and its Fraction  Produce

Example  1-1: Machi  Preparation of extract

Machi Hyun was washed with water, dried in the shade, and powdered with Waring brand. 12 kg of powdered mashin was added to 80 liters of ethanol, and the mixture was extracted with cold water for 7 days at room temperature. The mixture was filtered under reduced pressure through a filter paper (Wattsman, USA), and then the ethanolic solvent was removed at room temperature using a vacuum rotary evaporator. To obtain 612 g of crude extract.

Example 1-2: Portulaca Oleracea Preparation of the fraction and the first fraction was purified by column chromatography (fraction)

In order to separate the active fractions from the crude extract, the crude extract was suspended in 1 L of water, and the same amount of hexane was added to the mixture. The fractions were repeated three times to obtain 1 L of the water-soluble fraction and 3 L of the hexane-soluble fraction . The hexane-soluble fraction was concentrated under reduced pressure to obtain 176 g of hexane soluble extract.

Ethyl acetate was added to and mixed with 1 L of the water-soluble fraction, and this procedure was repeated three times to obtain 1 L of the water-soluble fraction and 3 L of the ethyl acetate soluble fraction. The ethyl acetate soluble fraction was concentrated under reduced pressure to obtain 381 g of the ethyl acetate- And the remaining water-soluble fraction was concentrated under reduced pressure to obtain 201 g, which was used as a water fraction.

The ethyl acetate fractions were subjected to silica gel column chromatography using hexane, ethyl acetate, and a mixed solvent of ethyl acetate and methanol. A solvent of hexane and ethyl acetate was used in a ratio of 100: 0 to 0: 100 (v / v), and a solvent of ethyl acetate and methanol was used in a ratio of 100: 0 to 0: 100 (v / v). Each elution solvent was analyzed by thin layer chromatography (TLC) and then divided into 12 small fractions, which were concentrated under reduced pressure and used as a primary column chromatography fraction of ethyl acetate layer.

Experimental Example  One: Machi  Extract, objection Fraction  And primary column  ≪ RTI ID = 0.0 > IL-6 < / RTI > STAT3 Of transcriptional activity

The following experiment was conducted to measure the inhibitory effect of STAT3 on the IL-6-induced IL-6 induction of the extracts of Liliaceae, its fractions and primary column chromatography fractions.

Experimental Example  1-1: Preparation of transformants

PStat3-Luc and pcDNA3.1 (+) (Clontech laboratories, Palo Alto, Calif.) Expressing luciferase by STAT3 in Hep3B cells (ATCC HB-8064) were transfected with lipofectamine plus (Invitrogen, Carlsbad, . ≪ / RTI > Two days after the transfection, hygromycin was treated (100 μg / ml) to obtain a clone in which luciferase was stably expressed. Whether the luciferase was stably expressed in this clone was confirmed through luciferase assays.

Experimental Example  1-2: IL -6 Reactivity STAT3  Reporter gene testing

The transfected cells were serum-starvated with DMEM medium (GIBCO 119950965), treated with 1 ng / ml IL-6 (R & D system, USA) for 12 hours .

1: negative control group (untreated group);

2: positive control (10 ng / ml of IL-6);

3: Extracts and fractions (10, 30 and 60 μg / ml), primary column chromatography fractions (10 and 30 μg / ml)

The cells were washed with PBS and mixed with 50 μl luciferase assay system (Promega, USA) for 20 minutes. Then, 30 to 100 μl of luciferase assay system (promega, USA) was added The degree of color development was measured with a luminometer (EG & G BERTHOLD, USA) within 5 minutes.

As a result, the hatching ethanol extract, hexane and ethyl acetate fractions, and the first column chromatography fraction showed an effect of inhibiting STAT3 luciferase activity in a concentration-dependent manner (FIGS. 1 and 2). Specifically, in the case of the hatching ethanol extract, 43.5%, 84.2% and 98.1% inhibitory effect was obtained at 10 μg / ml, 30 μg / ml and 60 μg / ml, The inhibitory effect was 90.1%, 99.3% and 100.7% at 30 ㎍ / ㎖ and 60 ㎍ / ㎖ respectively, and 66.5% at 10 ㎍ / ㎖, 30 ㎍ / ㎖ and 60 ㎍ / ㎖ for the ethyl acetate fraction, respectively. , 98.8% and 101.5%, respectively (Fig. 1).

For the first column chromatography fraction of the ethyl acetate layer, hexane and ethyl acetate fraction 1 (100: 0 (v / v)) and fraction 2 (100: 1 (v / v) 98.3% and 102.4%, respectively. After that, no activity was observed in fraction 3 and fraction 4, but activity increased again from fraction 5 (10: 1 (v / v)). Particularly, fraction 9, which was a 100% fraction of ethyl acetate, showed complete inhibitory activity at both 10 μg / ml and 30 μg / ml concentrations (FIG. 2). Later, ethyl acetate and methanol fractions also showed STAT3 luciferase inhibitory activity.

Experimental Example  1-3: IL -6 induction Stat3  Phosphorylation inhibition activity assay

Hep3B and U266 cells were cultured in a 6-well plate at a density of 80% (confluency) at 5 × 10 ⁵ cells / well, exchanged with serum-free medium, and further cultured for 12 hours. Respectively.

1: negative control group (untreated group);

2: positive control (10 ng / ml of IL-6);

3: Extract (10, 30, 60 占 퐂 / ml)

4: Genestein treated group (100 [mu] M)

Then, 10 ng / ㎖ IL-6 was treated for 20 minutes 40㎕ lysis buffer after the reaction (pH 8, 20mM Tris-HCl , 137mM NaCl, 10% glycerol, 1% Triton X-100, 1mM Na 3 VO (13,000 g, 15 min) after lysing the cells using ₄ mM EDTA, 1 mM PMSF, 20 mM leupeptin, 20 mg / ml aprotonin; Sigma, USA) A supernatant liquid was obtained. Protein concentration was determined using a DC protein test kit (Bio-Rad, USA) and the protein was loaded onto 4-12% SDS polyacrylamide gel (SDS-PAGE) and electrophoresed at 175 mA for 2 hours. After electrophoresis, proteins of the gel were transferred to PVDF membrane (Westran S, pore size 0.2 mm; Whatman, USA) at 35 V for 90 minutes. The transferred membrane was blocked with T-TBS (50 mM Tri-HCl, pH 7.6, 150 mM NaCl, 0.2% Tween-20, 5% skim milk; Sigma, USA) for 1 hour at room temperature, 5 times. The membrane was treated with phospho-Stat3 (1: 1,000 dilution) polyclonal antibody as a primary antibody for 12 hours at 4 ° C. After washing 5 times with T-TBS, HRP-conjugated anti-mouse antibody (1: 5,000 dilution) was reacted with secondary antibody for 1 hour. The films were then washed with T-TBS and developed in a dark room using an ECL kit (Amersham, USA). The results are shown in Fig.

As a result, as shown in Fig. 3, the horseradish ethanol extract of the present invention exhibited IL-6-induced Stat3 phosphorylation inhibitory activity, which was consistent with the results of luciferase assays confirmed in Figs. 1 and 2 above.

Thus, the above results show that the present invention can very effectively inhibit IL-6-induced STAT3 activity by the ethanol-extract, hexane and ethyl acetate fractions and the first column chromatography fraction of the present invention. Fractions and the like may be effectively used in IL-6 mediated diseases.

Experimental Example  2: Machi  Extract and its Fraction  Cytotoxicity confirmation experiment

The cytotoxicity confirmation experiment was carried out in order to confirm whether the extract of the present invention and its fractions can exhibit cytotoxicity. Hep3B cells were seeded at 2 × 10 ⁴ cells / well in a 96-well plate, cultured for 24 hours, and then replaced with fresh medium. The WST-1 reagent was treated with 10 μl / well of the WST-1 reagent and cultured for 4 hours in each well. The cells were treated with the ethanol extract of Marchia, hexane, ethyl acetate and water fractions at a concentration of 25 μg / ml and 50 μg / do. Absorbance was measured at 450 nm using a 96-well plate reader.

As a result, the horseradish ethanol extract, hexane, ethyl acetate and water fractions of the present invention showed a survival rate of 83% or more at a concentration of 25 μg / ml and 50 μg / ml, and the extract and fraction of the present invention showed no cytotoxicity (Fig. 4).

Experimental Example  3: Machi  The extract and Fraction RANKL -Induced osteoclast differentiation inhibition effect

In Experimental Example 1, it was confirmed that the mashin extract of the present invention and its fractions can effectively inhibit the activity of STAT3 induced by IL-6, and induced by RANKL, which is known to be mainly involved in the activity of STAT3 In order to examine whether osteoclast differentiation can be inhibited, the following experiment was conducted.

Experimental Example  3-1: RANKL  Induction osteoclast differentiation inhibitory effect test

Specifically, bone marrow cells were obtained by separating the thigh bone and the shin bone of 5-week-old mice and washing the bone lumen space with a 1 cc syringe. Separated bone marrow cells were cultured in a-minimal essential medium (a-MEM) medium containing 10% FBS, antibiotics and M-CSF (30 ng / ml) for 3 days. Three days later, adherent cells were used as bone marrow macrophages (BMM). The mesenchymal cells were cultured by the addition of M-CSF (30 ng / ml) and RANKL (50 ng / ml) and treated with gangrene extracts (50, 25 and 10 μg / ml) and fractions (50 μg / ml). After 4 days, the cultured cells were stained with TRAP solution (Sigma Aldrich, USA), and the red stained cells were identified as differentiated osteoclasts. The results are shown in FIGS. 5 and 6.

As a result, the horseradish ethanol extract, hexane and ethyl acetate fractions of the present invention effectively inhibited osteoclast differentiation induced by RANKL (FIGS. 5 and 6).

Experimental Example  3-2: RANKL Of the genes that play an important role in osteoclast differentiation induced by mRNA  Expression analysis

RANKL binds to RANK in the cell membrane and then through the intracellular signaling pathway, promotes the expression of transcription factors c-Fos and NFATc1 and induces the expression of TRAP gene, an indicator of osteoclast. MRNA expression of c-Fos, NFATc1, TRAP, and OSCAR was quantitated by real-time PCR in BMM stimulated with RANKL. BMM was prepared as in Experimental Example 2-1, followed by pretreatment with a hatching ethanol extract (50 μg / ml) for 1 hour, adding RANKL (100 ng / ml), and culturing for 12, 24 and 48 hours. After incubation, RNA was isolated from each cell with TRIzol (Invitrogen) solution, and cDNA was synthesized using 1 μg of the separated RNA. The synthesized cDNA was amplified by PCR using the following primers.

gene order SEQ ID NO: c-Fos
 sense 5'-CTGGTGCAGCCCACTCTGGTC-3 ' One antisense 5'-CTTTCAGCAGATTGGCAATCTC-3 ' 2 NFATc1
 sense 5'-CAACGCCCTGACCACCGATAG-3 ' 3 antisense 5'-GGCTGCCTTCCGTCTCATAGT-3 ' 4 OSCAR
 sense 5'-CTGCTGGTAACGGATCAGCTCCCCAGA-3 ' 5 antisense 5'-CCAAGGAGCCAGAACCTTCGAAACT-3 ' 6 TRAP
sense 5'-ACTTCCCCAGCCCTTACTAC-3 ' 7 antisense 5'-TCAGCACATAGCCCACACCG-3 ' 8 GAPDH
sense 5'-ACCACAGTCCATGCCATCAC-3 ' 9 antisense 5'-TCCACCACCCTGTTGCTGTA-3 ' 10

As a result of the PCR analysis, it was found that the horseradish ethanol extract of the present invention effectively suppressed mRNA expression of c-Fos, NFATc1, TRAP, and OSCAR, which are important genes for osteoclast differentiation (Fig. 7). In particular, the ethanol extract of mashhyun effectively inhibited mRNA expression of TRAP and OSCAR.

Experimental Example  3-3: RANKL Analysis of the lower signal transduction pathway; MAPK , AKT , And IκB phosphorylation measurement

BMM was prepared as described in Experimental Example 3-1, and then the medium was replaced with serum-free medium. After 3 hours of incubation in serum-free medium, the ethanolic extract of MCHC was pretreated for 1 hour at each concentration. Then, RANKL (100 ng / ml) was added and stimulated for 15 minutes. Then, each cell was collected and phosphorylation of each protein was measured in the same manner as in Experimental Example 1-3.

As a result, the ethanol extract of mashin inhibited the phosphorylation of AKT and IκB induced by RANKL but did not affect the phosphorylation activity of MAPK (p38, JNK, ERK) (FIG. 8).

Experimental Example  3-4: bone resorption test

Specifically, the osteoblast cells were separated from calvaria at 1 day old mice and added with 0.1% collagenase and 0.2% dispase, and the extracted cells were treated with 10% FBS and penicillin / streptomycin) for 3 days. Bone marrow cells and osteoblasts were added to collagen-coated 90-mm culture dishes and treated with vitamin D ₃ (10 ^-8 M) and PGE2 (10 ^-6 M) for 6 days. After 6 days, the adherent osteoclasts were removed using collagenase and then placed in a 48-well plate coated with hydroxyapatite and incubated for 1 hour. After incubation for 1 hour, trypsin was removed to remove osteoblast cells, pure osteoclasts were treated with RANKL (100 ng / ml), and the ganoderma ethanol extract was treated at different concentrations for 24 hours. The osteoclasts were removed by treatment with 0.1% Triton X-100 and photographed with an optical microscope. Quantification of the bone resorption region was performed using Image Pro-plus program version, version. 4.0 (Media Cybernetics).

As a result, it was found that the marine ethanol extract of the present invention effectively inhibited the osteoclastic bone resorption action at a concentration of 50 μg / ml (FIG. 9).

Experimental Example  3-5: LPS - Effectiveness of osteoporosis treatment in an animal model of induced bone resorption

In order to confirm the therapeutic effect of osteoporosis of the mashin ethanol extract of the present invention, an experiment was conducted using an LPS-induced bone resorption mouse model having characteristics similar to human osteoporosis. Specifically, LPS (lipopolysaccharide) was dissolved in PBS and injected into the abdominal cavity of ICR mice at a concentration of 5 mg / kg daily for 4 days. The ethanol extract of Munchhenae was dissolved in PBS at 400 mg / kg and the mice were orally administered once daily for 8 days from the day before LPS injection. In the normal group and the control group, PBS was orally administered, PBS was administered in the normal group, and LPS was intraperitoneally injected in the control group. During the experimental period, the ethanol extract of Ganoderma lucidum did not show any toxic signal and no abnormality of mouse was observed. After the administration, the femur of the mouse was separated and bone analysis of the femur was performed using micro-CT (micro-CT). Bone analysis (BT / TV (%), bone thickness (Tb.Th), bone marrow (Tb.N), and landing area measurement (Tb.Sp) were performed.

As a result, as shown in Fig. 10, the group of the ethanolic extract of the present invention continuously administered orally and LPS injected showed the lowest values in the three indexes of the postural bone mass, the landing space area, and the postural bone marrow All of them showed the effect of treating osteoporosis.

These results demonstrate that the Ganoderma lucidum extract or its fractions effectively inhibit the IL-6 mediated signal transduction pathway and can effectively treat IL-6 mediated diseases. In particular, the osteoclast differentiation and bone resorption And thus it is possible to effectively treat a bone metabolic disease, which is a representative IL-6 mediated disease.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the spirit and scope of the present invention as defined by the appended claims and their equivalents.

<110> Korea Research Institute of Bioscience and Biotechnology <120> A pharmaceutical composition for prevention or treating IL-6-mediated disease, comprising extract or fraction of Portulaca oleracea L. <130> PA130875KR <150> KR 10-2012-0112090 <151> 2012-10-09 <160> 10 <170> Kopatentin 2.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer for c-Fos (sense) <400> 1 ctggtgcagc ccactctggt c 21 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer for c-Fos (antisense) <400> 2 ctttcagcag attggcaatc tc 22 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer for NFATc1 (sense) <400> 3 caacgccctg accaccgata g 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer for NFATc1 (antisense) <400> 4 ggctgccttc cgtctcatag t 21 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer for OSCAR (sense) <400> 5 ctgctggtaa cggatcagct ccccaga 27 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer for OSCAR (antisense) <400> 6 ccaaggagcc agaaccttcg aaact 25 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for TRAP (sense) <400> 7 acttccccag cccttactac 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for TRAP (antisense) <400> 8 tcagcacata gcccacaccg 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for GAPDH (sense) <400> 9 accacagtcc atgccatcac 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for GAPDH (antisense) <400> 10 tccaccaccc tgttgctgta 20

Claims (14)

(IL-6) (interleukin-6) selected from the group consisting of bone metabolic diseases, inflammatory bowel disease, Crohn's disease, and combinations thereof, comprising hexane or ethyl acetate fractions of an extract of Portulaca oleracea L. A pharmaceutical composition for the prevention or treatment of an infectious disease.
2. The composition of claim 1, wherein the composition has STAT3 (signal transducers and activators of transcription 3) inhibitory activity.
The composition of claim 1, wherein the composition has an osteoclast differentiation inhibitory activity induced by RANKL (Receptor activator of nuclear factor kappa-B ligand).
delete delete delete delete 2. The composition of claim 1, wherein the bone metabolic disease is selected from the group consisting of osteoporosis, arthritis, periodontal disease, fracture and Paget's disease.
delete delete (IL-6) (interleukin-6) selected from the group consisting of bone metabolic diseases, inflammatory bowel disease, Crohn's disease, and combinations thereof, comprising hexane or ethyl acetate fractions of an extract of Portulaca oleracea L. A food composition for improving mediastinal disorders.
(IL-6) (interleukin-6) selected from the group consisting of bone metabolic diseases, inflammatory bowel disease, Crohn's disease, and combinations thereof, comprising hexane or ethyl acetate fractions of an extract of Portulaca oleracea L. A quasi-drug composition for improving mediastinal disorders.
(IL-6) (interleukin-6) selected from the group consisting of bone metabolic diseases, inflammatory bowel disease, Crohn's disease, and combinations thereof, comprising hexane or ethyl acetate fractions of an extract of Portulaca oleracea L. Feed additives or drinking water additives for the improvement of mediating diseases.
(Interleukin-6) mediated disease selected from the group consisting of bone metabolic diseases, inflammatory bowel disease, Crohn's disease, and combinations thereof, comprising administering the composition of claim 1 to a subject other than a human. Way.

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