KR102006998B1 - A pharmaceutical composition for preventing or treating IL-6-mediated disease, comprising extract, fraction, or compounds derived from Ampelopsis brevipedunculata - Google Patents

Abstract

The present invention is a vine ( Ampelopsis) brevipedunculata ) extract, a fraction thereof, or a compound isolated therefrom or a pharmaceutically acceptable salt thereof as an active ingredient, a pharmaceutical composition for the prevention or treatment of IL-6 (interleukin-6) mediated diseases and the composition The present invention relates to a method for treating IL-6 mediated disease, comprising administering to a subject suspected of IL-6 mediated disease. In addition, the present invention includes a jasmine vine extract, fractions thereof, or a compound isolated therefrom or a pharmaceutically acceptable salt thereof as an active ingredient, a food composition for preventing or ameliorating IL-6 mediated diseases, quasi-drug compositions, It relates to feed additives and drinking water additives.

Description

A pharmaceutical composition for preventing or treating IL-6-mediated disease, comprising extract, a fraction thereof, or a compound isolated therefrom as an active ingredient fraction, or compounds derived from Ampelopsis brevipedunculata}

The present invention is a vine ( Ampelopsis brevipedunculata ) extract, a fraction thereof, or a compound isolated therefrom or a pharmaceutically acceptable salt thereof, as an active ingredient, a pharmaceutical composition for preventing or treating IL-6 (interleukin-6) mediated diseases, and the composition It relates to a method of treating an IL-6 mediated disease comprising administering to an individual suspected of an IL-6 mediated disease. In addition, the present invention is a food composition, quasi-drug composition for preventing or improving IL-6-mediated disease, comprising as an active ingredient a gorilla extract, a fraction thereof, or a compound isolated therefrom or a pharmaceutically acceptable salt thereof, It relates to feed additives and drinking water additives.

Interleukin-6 (IL-6) is a cytokine, also called B cell stimulating factor 2 (BSF2) or interferon-2 (IFN-2), and was found to be a differentiation factor involved in the activation of B lymphocytes. After that, it was discovered that it is a multifunctional cytokine that affects the functions of several cells. IL-6 transmits its biological activity via two types of proteins on the cell membrane. One is the IL-6 receptor, which is a protein to which IL-6 binds. The IL-6 receptor is expressed through cell membranes and is a membrane-bound protein having 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 the non-ligand-binding signal transmission. IL-6 and IL-6 receptors form an IL-6/IL-6 receptor complex, which then binds to gp130. After binding of these ligands and receptors, JAK2 (Janus Kinase 2) is activated by transphosphorylation in the cell. Activated JAK2 phosphorylates several tyrosine residues in the cytoplasmic domains of the receptor, which are signal transducers and activators of STAT3, which have SH2 or other phosphotyrosine binding motifs. It acts as a docking site for proteins in the cytoplasm, such as transcription 3). STAT3 bound to the cytoplasmic domain of the receptor is phosphorylated by JAK2 and then released from the receptor, and these activated STAT3s bind to each other in the cytoplasm to form a homodimer or heterodimer, and then the nucleus It goes inside and binds to the recognition sequence of the target gene to increase transcription.

Various studies have been conducted on the relationship between the signaling system induced by IL-6 and inflammatory diseases, autoimmune diseases, and osteoporosis. Accordingly, studies to suppress the signaling system induced by IL-6 for the treatment of inflammatory, autoimmune diseases and osteoporosis are actively being conducted, and currently, anti-IL-6 against inhibition of the signaling system of IL-6 It is most commonly known to use an R antibody (anti-IL-6 receptor antibody). Synovial cell growth inhibitor for rheumatoid arthritis using the anti-IL-6 R antibody (International Patent Publication No. 96/011020), plasmacytosis using anti-IL-6 R antibody, hyperimmune globulinemia, anemia, nephritis, Treatment of diseases caused by IL-6 products such as cachexia, rheumatoid arthritis, herder's disease, and vasculogenic nephritis, etc. have already been known (International Publication No. 96/012503). In addition, anti-IL-6R antibodies are sensitive T cell-related diseases such as multiple sclerosis, uveitis, chronic thyroiditis, delayed hypersensitivity and atopic dermatitis (International Patent Publication No. 98/042377), treatment of systemic erythematos, Treatment of Crohn's disease (International Patent Publication No. 99/047170), Pancreatitis (International Patent Publication No. 00/010607), psoriasis treatment (International Patent Publication No. 02/034292) and juvenile chronic arthritis It has been reported that it can be applied to treatment (International Patent Publication No. 02/080969). However, in the case of the anti-IL-6 R antibody, when introduced into an individual, it may have an epitope that is a part that can be recognized as a foreign protein, and thus, when used as a therapeutic agent, it still has a disadvantage of acting as immunogenicity. In order to solve the above problems, many studies have been conducted to develop therapeutic agents for diseases mediated by IL-6 using small molecule compounds rather than proteins that are not recognized by the immune system.

Also, related to cancer, the IL-6 signaling system is closely related to its intermediate mediator, STAT3 (signal transducers and activators of transcription 3). STAT3 has been reported to be involved in several forms of cancer, including myeloma, breast carcinoma, prostate cancer, brain tumor, head and neck carcinoma, melanoma, leukemia and lymphoma, especially chronic myelogenous leukemia and multiple myeloma (Niu, et al., Cancer Res. ., 1999, 59, 5059-5063). Cells derived from both murine and human prostate cancer have been found to have structurally activated STAT3, and STAT3 has some acute leukemia (Gouilleux-Gruart, V. et al., Leuk. Lymphoma, 1997, 28, 83-88) and T It has been found to be structurally activated in cellular lymphoma (Yu, CL et al., J. Immunol., 1997, 159, 5206-5210). Interestingly, STAT3 has been shown to be structurally phosphorylated on serine residues in chronic lymphocytic leukemia (Frank, D.A., et al., J. Clin. Invest., 1997, 100, 3140-3148). STAT3 has been found to be structurally active in myeloma tumor cells, both in myeloma mononuclear cells and cultures from patients with multiple myeloma. These cells are resistant to Fas-mediated apoptosis and express high levels of Bcl-xL. STAT3 signaling has been shown to be essential for the survival of myeloma tumor cells by conferring resistance to apoptosis (Catlett-Falcone, R. et al., Immunity, 1999, 10, 105-115). On the other hand, in recent years, IL-6 is secreted ideally in patients with Ras-induced cancer including pancreatic cancer, and by removing IL-6, the growth of tumor cells and angiogenesis by Ras are inhibited, as well as the size of the tumor is reduced. Has been reported (Brooke Ancrile et al., Gene & Development, 2007, 21, 1714-1719). In addition, as it was found that STAT3 is activated by overexpression of IL-6 in EGFR-mutated lung adenocarcinoma, the gp130/JAK/STAT3 pathway by IL-6 is emerging as a new target in chemotherapy (Sizhi Paul Gao et al., J. Clin. Invest. 2007, 117, 38463856).

In addition, it is known that STAT3 plays a role as another important transcription factor different from NF-κB in autoimmune diseases including rheumatoid arthritis or osteoporosis. In particular, it is well known that STAT3 is activated by the cytokine IL-6, and it is also known to be activated by epidermal growth factor (EGF). Recently, many studies have been reported on the association between osteoporosis and IL-6, and in particular, it has been proven through animal models that STAT3 activity induced by IL-6 plays an important role in the differentiation of osteoclasts and bone formation. (Bone 2006, 39, 505-512), in the case of STAT3-deficient mice, bone mineral density and bone volume were decreased, and an increase in the number of osteoclasts, which negatively affects osteoporosis, was observed (BBRC 2005). , 328, 800-807). On the other hand, when madindoline A, a compound that inhibits IL-6-induced phosphorylation of STAT3, was treated in OVX mice, an animal model for postmenopausal osteoporosis experiments, the activity of inhibiting bone resorption was shown. (PNAS, 2002, 99(23), 14728-14733), in the IL-6-/- mouse model, no changes such as bone loss due to estrogen depletion and an increase in the number of osteoclast precursors were observed (EMBO J 1994, 13, 1189-1196). These facts indicate that IL-6 plays an important role in bone formation and resorption, in particular, through phosphorylation of STAT3.

Accordingly, the present inventors made intensive efforts to find a natural product capable of treating IL-6-mediated diseases by targeting the STAT3 pathway activated by IL-6. As a result, the extract and fractions thereof inhibited the transcriptional activity and phosphorylation of STAT3. Thus, it was confirmed that it has an excellent activity that effectively inhibits the signaling pathway mediated by IL-6 and, in particular, inhibits the differentiation of osteoclasts, and that it can be used for the prevention or treatment of IL-6-mediated diseases. I did. In addition, an effective compound having an activity of effectively inhibiting the IL-6-mediated signaling pathway and inhibiting the differentiation of osteoclasts has been identified from the extract of Asteraceae extract and applied to the treatment of IL-6-mediated diseases including bone metabolic diseases. It was confirmed that it could be done, and the present invention was completed.

One object of the present invention is a vine ( Ampelopsis brevipedunculata ) To provide a pharmaceutical composition for the prevention or treatment of IL-6 (interleukin-6) mediated disease, comprising an extract or a fraction thereof as an active ingredient.

Another object of the present invention is to provide a food composition for preventing or improving IL-6-mediated diseases, a quasi-drug composition, a feed additive and a drinking water additive, comprising an extract or a fraction thereof as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating IL-6 mediated diseases, comprising a compound represented by any one of the following Formulas 1 to 3, or a pharmaceutically acceptable salt thereof, as an active ingredient It is to do.

[Formula 1]

[Formula 2]

[Formula 3]

Another object of the present invention is a food composition, quasi-drug composition for preventing or improving IL-6-mediated diseases, comprising a compound represented by any one of Formulas 1 to 3, or a pharmaceutically acceptable salt thereof, as an active ingredient , To provide feed additives and drinking water additives.

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

In some embodiments for achieving the above object, the present invention is a pharmaceutical for preventing or treating containing ampelopsis brevipedunculata vines (Ampelopsis brevipedunculata) extract or fractions thereof as an active ingredient, IL-6 (interleukin-6 ) mediated diseases The composition is provided.

In the present invention, the term " Ampelopsis brevipedunculata )" is a vine deciduous shrub belonging to the genus Grapeaceae coccinus . The coccyx is known to have hemostatic action, antibacterial action, antioxidant action, etc. in its pharmacological activity, but the coriander extract or its fractions are particularly inflammatory diseases, There is no known use for treatment in bone metabolic diseases, atopy and cancer, and was first identified by the present inventors In the present invention, the honeysuckle can be purchased commercially, or harvested or cultivated in nature can be used. have.

In the present invention, the term "black vine extract" refers to an extract obtained by extracting black vine. _{The extract is a carbon number of 1 (C 1} ) to 6 (C) such as water, methanol, ethanol, butanol, etc. in a volume of about 2 to 20 times, preferably about 3 to 5 times the dry weight of the crushed blackberry vine. ₆ ) of an alcohol polar solvent, preferably a lower alcohol polar solvent having 1 to 4 carbon atoms, or a mixed solvent having a mixing ratio of about 1:0.1 to 1:10 thereof, as an elution solvent, and the extraction temperature is 20°C to At 100° C., preferably at room temperature, the extraction period can be extracted using an extraction method such as hot water extraction, cold needle extraction, reflux cooling extraction, or ultrasonic extraction for about 12 hours to 4 days, preferably 3 days, but IL -6 Any method of extracting a substance having therapeutic activity for a mediated disease can be used without limitation. Preferably, extraction is carried out under reduced pressure after 1 to 5 consecutive extractions by cold sedimentation, and the filtered extract is concentrated under reduced pressure at 20°C to 100°C with a vacuum rotary concentrator, more preferably at room temperature, and water, lower alcohol or a mixed solvent thereof Although it may be a result obtained by obtaining the crude extract of black stalk vine that can be used for, the extract is not limited thereto, as long as it can exhibit the therapeutic effect of the IL-6-mediated disease of the present invention, and the extract, a diluted solution or a concentrate of the extract, or an extract may be used. It includes all forms of dried products obtained by drying, or preparations or purified products thereof. The anteater extract can be extracted from various organs of natural, hybrid, and variant plants, and can be extracted from, for example, roots, above-ground parts, stems, leaves, flowers, trunks of fruits, shells of fruits, as well as plant tissue cultures. Do. In one embodiment of the present invention, ethanol was added to the stem of the powdered blackberry vine, and cold sediment was extracted for 7 days (Example 1).

In the present invention, the term "fraction" means a product obtained by a fractionation method for separating a specific component or a specific group from a mixture containing various constituents. In the fractions of the vines of the present invention, a polar solvent fraction and a fraction of a non-polar solvent can be obtained, respectively, by suspending the vine extract in water and then fractionating using a non-polar solvent such as hexane and ethyl acetate. Specifically, after suspending the crude blackberry vine extract in distilled water, about 1 to 100 times, preferably about 1 to 5 times the volume of the suspension, non-polar solvents such as hexane and ethyl acetate are added once to 10 times, preferably Can be obtained by extracting and separating the non-polar solvent soluble layer over 2 to 5 times. In addition, it is also possible to perform a conventional fractionation process (Harborne JB Phytochemical methods: A guide to modern techniques of plant analysis, 3rd Ed. p6-7, 1998). More specifically, after suspending the crude blackberry extract in water, continuous extraction using the same amount of n -hexane and ethylacetate solvent to obtain each solvent-soluble extract of the blackberry vine, more specifically, After the extract is suspended in water, the same amount of n -hexane is mixed and fractionated to obtain an n- hexane-soluble fraction and a water-soluble fraction, and ethyl acetate is added to the water-soluble fraction to obtain an ethyl acetate-soluble fraction and a water-soluble fraction. Fractions can be obtained. In one embodiment of the present invention, the crude extract of blackberry vine was suspended in water to obtain a water fraction, a hexane fraction, and an ethyl acetate fraction (Example 1-2).

In addition, the extract or a fraction thereof may include a compound represented by the following formulas 1 to 3 as a representative active ingredient.

[Formula 1]

[Formula 2]

[Formula 3]

In one embodiment of the present invention, three kinds of effective compounds that effectively block the IL-6 signaling pathway were isolated from the extract or fractions thereof.

Specifically, the three effective compounds are ethyl gallate of formula 1, gallic acid of formula 2, and aromadendrin of formula 3, ethyl gallate is ethyl 3,4, 5-trihydroxybenzoate (ethyl 3,4,5-trihydroxybenzoate), gallic acid 4-hydroxy-3-methoxybenzoic acid, aromadendrine 3,5 ,7-trihydroxy-2-(4-hydroxyphenyl)chroman-4-one (3,5,7-trihydroxy-2-(4-hydroxyphenyl)chroman-4-one).

The method for separating the compounds may be carried out as follows.

Specifically, the compound of Formulas 1 to 3 may be obtained by separating and purifying the compound by silica gel chromatography from the hexane fraction and ethyl acetate fraction. When performing a silica gel chromatography for the compound separation, mobile phase n - in hexane-ethyl acetate, chloroform and acetone, a mixed solvent and chromatography, preferably using methanol, and add n - - hexane, n-hexane and acetone mixture A solvent can be used. The volume ratio of the n -hexane·ethylacetate mixed solvent used at this time is preferably 50:1 to 1:5 by volume, and in the case of a chloroform·acetone mixed solvent, 150:1 to 1:4 by volume is preferable. The chromatography may be performed once to several times until a single compound is purified, and concentration and recrystallization may be performed if necessary.

The three effective compounds of the present invention effectively inhibit STAT3 activation by IL-6 treatment (FIG. 4), and thus can be applied to the prevention or treatment of IL-6 mediated diseases. In addition, the three effective compounds effectively inhibit the differentiation of osteoclasts (FIG. 11), and thus have an effect that can be applied to the prevention or treatment of bone metabolic diseases including osteoporosis.

The extract, a fraction thereof, and the three compounds isolated therefrom of the present invention exhibit inhibitory activities of STAT3 (signal transducers and activators of transcription 3) and JAK2 (Janus kinase 2), and prevent IL-6-mediated diseases. Or it can be effectively used for treatment.

In the present invention, the term "IL-6-mediated disease" is a generic term for a disease to be invented by activation of the signaling pathway by IL-6. Specifically, as JAK2 and STAT3 are phosphorylated and activated by IL-6 It means an onset disease. Specifically, when IL-6 binds to receptors such as gp130 and gp80, JAK2 is phosphorylated and activated, which in turn leads to phosphorylation of the STAT3 protein, thereby activating the STAT3 protein. Activated STAT3 protein can lead to the onset or progression of IL-6-mediated diseases by entering the nucleus and expressing the target gene. For the purposes of the present invention, the IL-6 mediated disease includes without limitation any disease that can be prevented or treated by the extract or fractions thereof of the present invention, such as inflammatory disease, bone metabolic disease, atopy or cancer. However, it is not limited thereto.

In the present invention, the term "inflammatory disease" refers to a disease whose main lesion is inflammation, and for the purposes of the present invention may mean an inflammatory disease mediated by STAT3 activated by IL-6. Examples of such inflammatory diseases include allergy, pheocytosis, hyperimmune globulinemia, anemia, nephritis, cachexia, herder's disease, vasculitis, multiple sclerosis, uveitis, chronic thyroiditis, delayed hypersensitivity, contact dermatitis, systemic erythematos, and Crohn Disease, psoriasis, juvenile idiopathic atrophy, diabetes or Alzheimer's, but is not limited thereto. Since the STAT3 protein activated in inflammatory diseases acts as a major transcription factor, leading to the onset and progression of inflammatory diseases, the composition of the present invention that effectively inhibits the activation of STAT3 protein can be usefully used in the prevention and treatment of inflammatory diseases. have.

In the present invention, the term "bone metabolic disease" refers to a bone-related disease caused by an imbalance of osteoblasts and osteoclasts. Examples of such disorders are pathological bone diseases such as osteoprosis, arthritis, periodontal disease, fractures, or Paget disease due to excessive bone resorption of osteoclasts, which promotes bone destruction. The disease includes, but is not limited to the bone metabolic disease.

Among them, "osteoporosis" refers to a condition in which the amount of bone is decreased and the strength of the bone is weakened due to qualitative changes, so that a fracture is likely to occur. Bone protects various organs in the human body and functions as a reservoir to store necessary substances in the body, such as calcium, and balance between osteoclasts that break down bone and osteoblasts that produce bones in bone tissue. Subsequently, homeostasis is maintained. In the case of osteoporosis, the balance of the two cell activities is disrupted, causing excessive bone destruction by osteoclasts, leading to onset and progression. The major molecule in osteoporosis is RANKL (receptor activator of nuclear factor kappa-B ligand), and RANKL binds to its receptor and activates various transcription factors to promote the differentiation of osteoclasts. It is known to promote osteoclast differentiation mediated by (Kim K et al., J Immunol. 2007 May 1;178(9):5588-94.). Therefore, a substance capable of reducing the destruction of bone by osteoclasts by inhibiting the signal transduction activated by IL-6 and inhibiting the differentiation of osteoclasts can be used as a therapeutic agent for osteoporosis. The extract, a fraction thereof, or a compound of Formulas 1 to 3 isolated therefrom of the present invention effectively inhibits the inhibition of osteoclast differentiation induced by RANKL, and thus can be usefully used in the prevention or treatment of osteoporosis.

The "arthritis" refers to a joint disease involving inflammation in one or more joint regions. The common form of arthritis is osteoarthritis, in which inflammation and pain occur due to damage to the bones and ligaments constituting the joint due to gradual damage or degenerative changes in cartilage reporting the joint. For the purposes of the present invention, the arthritis is a disease involving bone loss in the joint region, and the type is not particularly limited, but may include osteoarthritis or rheumatoid arthritis.

The "periodontal disease" refers to an inflammatory state of the tooth supporting tissue caused by bacteria, and can be separated into gingivitis and periodontitis. The cause of the disease is the formation of a bacterial film when oral bacteria hit due to poor oral hygiene. The tooth surface bacterial film refers to a cluster of bacteria that has grown after bacteria adhere to the tooth surface by using a sticky substance in saliva as an adhesive. If left untreated, the bacterial membrane of the tooth may become inflamed, sometimes bleeding from the gums and bad breath. This symptom is called gingivitis. As gingivitis progresses further, the gap between the teeth and the gums becomes deeper, resulting in a periodontal sac, where bacteria that cause periodontal disease multiply, resulting in periodontitis. When periodontitis progresses, the gums bleed and swell even with weak stimuli, such as brushing, and often turn into acute inflammation, causing pain. This inflammation decreases the function of making bones, and increases the function of absorbing the bones, so that the alveolar bone is gradually lowered and the alveolar bone is destroyed and eventually the teeth are lost.

The "fracture" refers to a state in which the continuity of a bone or epiphysis plate or joint surface is abnormally broken, and refers to a fracture of a bone. The causes of fractures include trauma such as traffic accidents, safety accidents occurring in industrial disorders, bone changes due to diseases such as osteoporosis, bone cancer, and metabolic dystrophy, and repetitive stress on bones due to sports or loads. In addition, the fracture state is based on the fracture line (the line along the end of the bone caused by bone cutting), crack fracture, greenstick fracture, transverse fracture, sagittal fracture, spiral fracture, segmental fracture, comminuted fracture, avulsion fracture. , Compression fracture, depression fracture, etc.

The "Paget's disease" refers to a localized bone disease in which bone remodeling is excessively promoted and the skeletal system in a wide area is invaded. The pathological mechanism of Paget's disease is known to be a combination of an excessive increase in bone resorption by osteoclasts, which have a function to clean the bones, and an increase in new bone formation by osteoblasts, which have a function of making bones, as a reward. In addition, bones newly formed in Bone Paget's disease are structurally disordered and are known to be very susceptible to bone deformation and fracture.

In the prophylaxis or treatment of bone metabolic diseases as described above, the extract, a fraction thereof, or a compound isolated therefrom of the present invention inhibits the differentiation of osteoclasts and inhibits the bone resorption of osteoclasts. It can bring a prophylactic or therapeutic effect against diseases.

In the present invention, the term "atopy" is a kind of skin disease, and is a disease accompanied by swelling of the skin, eczema, and itching. It is known that both environmental and genetic factors are involved in the onset of atopy, but the exact pathogenesis has not been identified. The atopy may have symptoms of recognizing an antigen from a protein derived from keratinocytes or endothelial cells and causing an immune response, but is not limited thereto. According to previously reported, atopy is characterized by having an autoantibody against CD28, and the CD28 autoantibody is presented as a feature that can not only activate T cells, but also differentiate it from atopy and other inflammatory skin diseases. (Clin Exp Immunol. 2006 Nov;146(2):262-9.). In recent years, atopy is frequently onset not only in children but also in adults, and its prevention or treatment has become an important social problem, but its cause has not been clearly identified. In addition, there is a problem of side effects as well as ineffectiveness of the existing steroid preparations, and thus there is a need to develop a safe atopy therapeutic agent that is excellent in effect and can be used for children as well. The composition of the present invention is derived from a natural product and has a safe advantage to be applied to infants and toddlers compared to steroid preparations. In addition, in the composition of the present invention, atopic vine extract or a fraction thereof included for the prevention or treatment of atopy blocks the IL-6-mediated pathway, resulting in atopic dermatitis including edema by the IL-6-mediated signaling pathway. In order to effectively treat atopic dermatitis by mitigating various inflammatory phenomena, it may be obtained by extracting from the stem portion of the vine, but is not limited thereto.

In the present invention, the term "cancer" includes without limitation any cancer that can be treated by the composition of the present invention, such as pancreatic cancer, breast cancer, prostate cancer, brain tumor, head and neck carcinoma, melanoma, myeloma, leukemia, lymphoma, liver cancer , Stomach cancer, colon cancer, bone cancer, uterine cancer, ovarian cancer, rectal cancer, esophageal cancer, small intestine cancer, anal muscle cancer, colon cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, bladder cancer, kidney cancer , Ureteral cancer, renal cell carcinoma, renal pelvic carcinoma or central nervous system tumor, but is not limited thereto.

In the present invention, the term "prevention" refers to all actions of inhibiting or delaying the onset of IL-6-mediated disease by administration of the composition, and "treatment" refers to IL-6-mediated disease by administration of the composition. It refers to any action in which the symptoms caused by or are advantageously changed.

The pharmaceutical composition comprising the extract or fractions thereof of the present invention may further include a suitable carrier, excipient, or diluent commonly used in the preparation of a pharmaceutical composition. At this time, the content of the extract or fractions thereof included in the composition is not particularly limited thereto, but may include 0.0001% by weight to 10% by weight, preferably 0.001% by weight to 1% by weight based on the total weight of the composition. have.

The pharmaceutical composition is any selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. It may have a single dosage form, and may be various oral or parenteral dosage forms. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations include at least one excipient in one or more compounds, such as starch, calcium carbonate, sucrose, or lactose ( lactose), gelatin, etc. In addition, in addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, syrups, etc., but may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to water and liquid paraffin, which are commonly used simple diluents. have. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

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

In the present invention, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type and severity of the individual, age, sex, and disease. It can be determined according to the type, activity of the drug, sensitivity to the drug, the time of administration, the route of administration and the rate of excretion, the duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And can be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all of the above factors, and can be easily determined by a person skilled in the art. The preferred dosage of the composition of the present invention varies depending on the condition and weight of the patient, the degree of the disease, the form of the drug, the route of administration and the duration, but for a desirable effect, the extract or fraction thereof of the present invention is 0.0001 to 100 mg per day. It is good to administer at /kg, preferably 0.001 to 100mg/kg. Administration may be administered once a day, or may be divided several times. The composition can be administered to various mammals such as mice, livestock, humans, etc. by various routes, and the mode of administration includes without limitation, if it is a conventional method in the art, for example, oral, rectal or intravenous, muscle, It can be administered by subcutaneous, intrauterine dura mater or intracerebrovascular injection.

In addition, the pharmaceutical composition of the present invention can be used in the form of veterinary drugs as well as pharmaceuticals applied to humans.

In one embodiment of the present invention, ethanol extract of vines; Its hexane, ethyl acetate and water fractions; And it was confirmed that the chromatographic fraction for the hexane fraction or the ethyl acetate fraction effectively inhibited the STAT3 activity induced by IL-6 (FIGS. 1 to 3 ). Particularly, the ethanol extract of the blackberry vine showed a result of inhibiting the phosphorylation of STAT3 and the phosphorylation of JAK2 in a concentration-dependent manner (Figs. 5 and 6), so that the ethanol extract and its fractions of the blackberry vine were used to treat diseases mediated by IL-6. It was confirmed that it can be usefully used for prevention or treatment. In addition, it was confirmed that the ethanol extract of the present invention effectively inhibits the differentiation of osteoclasts induced by RANKL (FIG. 7). Osteoclasts absorb bone and induce bone diseases such as osteoporosis. In particular, it was confirmed that the number of polynuclear osteoclasts was significantly reduced by the Ethanol extract (Fig. 7B), and it was confirmed that there is no cytotoxicity at the concentration of the E. kelp extract used (Fig. 7C). In addition, according to an embodiment of the present invention, expression of genes and proteins of c-Fos and NFATc1, which are transcription factors that are expressed by RANKL and play a key role in the differentiation of osteoclasts, are effectively inhibited by treatment with ethanol extract of A. It can be seen (Figs. 8A, 8B), and all of the gene expressions of OSCAR, TRAP, cathepsin K, and DC-STAMP, which are genes important for the differentiation of osteoclasts, were effectively suppressed by the ethanol extract of syllium syllium (Fig. In order to investigate the mechanism of action for inhibiting osteoclast differentiation by ethanol extract of Ethanol extract, phosphorylation of p38, AKT, ERK, and IκB, the sub-signaling mechanisms of RANKL, was measured. It inhibited the phosphorylation of IκB and did not affect the phosphorylation of AKT and ERK (FIG. 9). In addition, it could be seen that the ethanol extract of A. vines inhibited the formation of osteoclasts, as well as the bone resorption action of osteoclasts (FIG. 10). Therefore, the extract or a fraction thereof of the present invention can be usefully used in the prevention or treatment of IL-6 mediated diseases, particularly in the prevention or treatment of bone metabolic diseases including osteoporosis.

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

The vines, 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 improving IL-6-mediated diseases, preferably for the purpose of preventing or improving inflammatory diseases, bone metabolic diseases, atopy or cancer. have.

When the extract or a fraction thereof of the present invention is used as a food additive, the extract or a fraction thereof may be added as it is or may be used with other foods or ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient can be appropriately determined according to the intended use.

There is no particular limitation on the type of food of the present invention. Examples of foods to which the extract or fractions thereof can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages , Tea, drinks, alcoholic beverages, and vitamin complexes, and the like, may include all foods in the usual sense, and include foods used as feed for animals.

In addition to the above, the food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, colorants, pectic acids and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols. , May contain a carbon oxidizing agent used in carbonated beverages. In addition, it may contain flesh for the manufacture of natural fruit juice, fruit juice beverage and vegetable beverage. In addition, the food may be prepared in formulations such as tablets, granules, powders, capsules, liquid solutions, and pills according to known manufacturing methods. There is no particular limitation on other components other than including the extract of the blackberry vine according to the present invention or a fraction thereof as an active ingredient, and various conventional flavoring agents or natural carbohydrates may be included as additional components.

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

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

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

In the present invention, the term "quasi-drug" refers to fibers, rubber products, or the like, which are used for the purpose of treating, alleviating, treating, or preventing diseases of humans or animals, and their action on the human body is weak or does not directly act on the human body, and Or non-machine and similar, as an article corresponding to one of the preparations used for sterilization, insecticide, and similar purposes to prevent infection, and used for the purpose of diagnosing, treating, alleviating, treating or preventing diseases of humans or animals. It refers to items that are not instruments, machines, or devices among the items that are used, and items that are not instruments, machines, or devices among items used for the purpose of pharmacologically affecting the structure and function of humans or animals, and are intended for external use for skin and personal hygiene. Includes supplies.

When the extract or fraction thereof of the present invention is added to a quasi-drug composition for the purpose of preventing or improving IL-6-mediated disease, the extract or fraction may be added as it is or used with other quasi-drug components, and a conventional method It can be used appropriately according to. The mixing amount of the active ingredient can be appropriately determined according to the intended use.

The external preparation for skin is not particularly limited thereto, but may be preferably prepared and used in the form of an ointment, lotion, spray, patch, cream, powder, suspension, gel or gel. The personal hygiene products are not particularly limited thereto, but preferably soap, cosmetics, wet tissues, tissue paper, shampoo, skin cream, face cream, toothpaste, lipstick, perfume, makeup, foundation, cheek touch, mascara, eye shadow, sunscreen It can be a lotion, hair care product, air freshener gel, or cleaning gel. In addition, another example of the quasi-drug composition of the present invention is disinfectant cleaner, shower foam, gagrin, wet tissue, detergent soap, hand wash, humidifier filler, mask, ointment or filter filler.

In another aspect, the present invention provides a feed additive or a drinking water additive for preventing or improving IL-6-mediated diseases, comprising an extract or a fraction thereof as an active ingredient.

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

In the present invention, the term "feed additive" is a generic term for substances added in trace amounts to feed for nutritional or specific purposes, and in the present invention, it means substances added for the purpose of preventing or improving IL-6 mediated diseases do. Here, an animal is a concept including livestock and pets.

The feed additive of the present invention may further include a binder, emulsifier, preservative, etc. added to prevent quality degradation, and amino acid, vitamin, enzyme, probiotic, flavoring, non-protein nitrogen added to increase utility A compound, a silicate, a buffer, a colorant, an extractant, an oligosaccharide, etc. may be additionally included, and in addition, a feed mixture may be additionally included, but the present invention is not limited thereto.

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

As another aspect, the present invention is a pharmaceutical composition for the prevention or treatment of IL-6-mediated diseases, comprising a compound represented by any one of Formulas 1 to 3, or a pharmaceutically acceptable salt thereof, as an active ingredient Provides.

The compounds of Formulas 1 to 3, IL-6-mediated diseases, prevention or treatment, and pharmaceutical compositions are as described above.

In addition, the compounds of Formulas 1 to 3 may be separated and purified from natural products known in the art including them, or may be used by purchasing or synthesizing a commercially available compound, but is not limited thereto.

In addition, the compounds of Formulas 1 to 3 may be included in the composition of the present invention in the form of a pharmaceutically acceptable salt.

In the present invention, the term "pharmaceutically acceptable salt" refers to all salts of the compound of the present invention that the target individual is physiologically acceptable, and the biological activity of the compound does not cause serious irritation to the organism to which the compound is administered. It is preferable that it is a formulation of a compound that does not impair the properties and properties. The pharmaceutical salt is an acid forming a non-toxic acid addition salt containing a pharmaceutically acceptable anion, for example, an inorganic acid such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, tartaric acid, formic acid, Organic carbon acids such as citric acid, acetic acid, trichloroacetic acid, trichloroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, salicylic acid, etc., methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc. Acid addition salts formed by sulfonic acid and the like are included. For example, pharmaceutically acceptable carboxylate salts include metal salts or alkaline earth metal salts formed of lithium, sodium, potassium, calcium, magnesium, etc., amino acid salts such as lysine, arginine, guanidine, dicyclohexylamine, N Organic salts such as -methyl-D-glucamine, tris(hydroxymethyl) methylamine, diethanolamine, choline and triethylamine, and the like.

In another aspect, the present invention provides a food composition for preventing or improving IL-6-mediated diseases, comprising a compound represented by any one of Formulas 1 to 3, or a pharmaceutically acceptable salt thereof, as an active ingredient. to provide.

The compounds of Formulas 1 to 3, IL-6-mediated diseases, and food compositions are as described above.

In another aspect, the present invention provides a quasi-drug composition for preventing or improving IL-6-mediated diseases, comprising a compound represented by any one of Formulas 1 to 3, or a pharmaceutically acceptable salt thereof, as an active ingredient. to provide.

The compounds of Formulas 1 to 3, IL-6-mediated diseases, and quasi-drug compositions are as described above.

In another aspect, the present invention is a feed additive for preventing or improving IL-6-mediated diseases, comprising a compound represented by any one of Formulas 1 to 3, or a pharmaceutically acceptable salt thereof as an active ingredient, or Provides drinking water additives.

The compounds of Formulas 1 to 3, IL-6-mediated diseases, feed additives, and drinking water additives are as described above.

In another aspect, the present invention provides a composition comprising the extract, a fraction thereof, or a compound represented by any one of Formulas 1 to 3, or a pharmaceutically acceptable salt thereof, to an individual suspected of IL-6 mediated disease. It provides a method of treating an IL-6 mediated disease comprising the step of administering.

The composition, IL-6 mediated disease and treatment are as described above.

Specifically, the treatment method of the present invention includes administering the pharmaceutical composition in a pharmaceutically effective amount into an individual suspected of an IL-6 mediated disease. The individual refers to all mammals including dogs, cows, horses, rabbits, mice, rats, chickens, or humans, but the mammal of the present invention is not limited by the above examples. The pharmaceutical composition may be administered parenterally, subcutaneously, intraperitoneally, intraperitoneally and intranasally, and for local treatment, if necessary, may be administered by a suitable method including intralesional administration. The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and weight of the individual, the degree of disease, the form of the drug, the route and duration of administration, but may be appropriately selected by those skilled in the art.

Since the composition of the present invention effectively inhibits signaling by IL-6 and effectively inhibits the differentiation of osteoclasts, the composition of the present invention comprising the extract or a fraction thereof is an IL-6 mediated disease, such as an inflammatory disease. , It can be usefully used in the prevention or treatment of bone metabolic disease, atopy or cancer. In particular, the extract or a fraction thereof is derived from a natural product that has been used as a natural medicine for a long time, and thus has an advantage that can be used for the treatment of the disease without side effects.

1 is a diagram showing the activity of inhibiting the expression of IL-6-induced luciferase of ethanol extract, hexane, ethyl acetate, and water fractions.
FIG. 2 is a diagram showing the activity of inhibiting the expression of IL-6-induced luciferase in a primary column chromatography fraction of a hexane fraction of A. oleifera.
FIG. 3 is a diagram showing the activity of inhibiting the expression of IL-6-induced luciferase in the first column chromatography fraction of an ethylacetate fraction of A. oleifera.
FIG. 4 is a diagram showing the inhibitory activity of IL-6-induced luciferase expression of effective compounds (ethyl gallate, gallic acid, and aromadendrin) isolated from the extract of A.
Figure 5 is a diagram showing the IL-6-induced STAT3 phosphorylation inhibitory activity of ethanol extract of vines.
Figure 6 is a diagram showing the IL-6-induced JAK2 phosphorylation inhibitory activity of ethanol extract of blackberry vine.
Figure 7 is a diagram showing the RANKL-induced osteoclast differentiation inhibitory activity of ethanol extract of vines.
FIG. 8 is a diagram showing that the ethanol extract of Gamedica chinensis suppresses the expression of genes and proteins required for differentiation of RANKL-induced osteoclasts.
FIG. 9 is a diagram showing that the ethanol extract of A. vines inhibits the differentiation of osteoclasts by inhibiting the phosphorylation mechanisms of p38 and IκB, which are sub-signaling mechanisms of RANKL.
FIG. 10 is a diagram showing the activity of the ethanol extract of Gamerilla vine to inhibit bone resorption of osteoclasts.
11 is a diagram showing the RANKL-induced osteoclast differentiation inhibitory activity of the isolated effective compounds, ethyl gallate (EG), gallic acid (GA), and aromadendrine (AD).

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

Example 1: Preparation of extract and fractions thereof

Example 1-1: Preparation of extract of black stalk vine

After washing the stem part of the stalk with water and drying it in the shade, it was powdered with a Waring brand. 6kg of powdered blackberry vine was put in 60ℓ of ethanol, extracted with cold for 7 days at room temperature, filtered under reduced pressure with filter paper (Whatman, USA), and then the filtered extract was removed from the ethanol solvent at room temperature with a vacuum rotary concentrator, and the extracted As a residue, 250 g of a crude extract of blackberry vine was obtained.

Example 1-2: Preparation of a black honeysuckle fraction and a first column chromatography fraction

In order to separate the active fraction from the crude extract, the crude blackberry vine extract was suspended in 1 liter of water and then fractionated by adding the same amount of hexane and mixing, and this process was repeated 3 times to obtain 1 liter of a water-soluble fraction and 3 liter of a hexane-soluble fraction. Thereafter, the hexane-soluble fraction was concentrated under reduced pressure to obtain 32 g of a hexane-soluble extract. Ethyl acetate was added to 1 liter of the water-soluble fraction and fractionated, and this process was repeated three times to obtain 1 liter of a water-soluble fraction and 3 liter of an ethyl acetate-soluble fraction. The ethyl acetate-soluble fraction was concentrated under reduced pressure to obtain 75 g of ethyl acetate-soluble extract. Was obtained, and the remaining water-soluble fraction was concentrated under reduced pressure to obtain 100 g, which was used as a water fraction.

The hexane fraction was subjected to silica gel column chromatography using a mixed solvent of chloroform and methanol. At this time, a solvent of 50:1 to 0:100 (v/v) was used for chloroform and methanol. Each elution solvent was analyzed by thin-layer chromatography (TLC), divided into 10 small fractions, concentrated under reduced pressure, and used as a hexane layer first column chromatography fraction. Further, the ethyl acetate fraction was subjected to silica gel column chromatography using a mixed solvent of chloroform and methanol. At this time, a solvent of 50:1 to 0:100 (v/v) was used for chloroform and methanol. Each elution solvent was analyzed by TLC, divided into 13 small fractions, concentrated under reduced pressure, and the ethyl acetate layer was used as a first column chromatography fraction.

Example 2: Isolation and Structure Identification of Active Compounds from Extracts and Fractions thereof

Example 2-1: Separation and purification of an effective compound

A 107 g fraction obtained by combining the hexane fraction and the ethyl acetate fraction was subjected to silica gel column chromatography using a mixed solvent of hexane, ethyl acetate, chloroform and methanol. At this time, a mixed solvent ratio of hexane, ethyl acetate, chloroform and methanol was 100: 0: 0: 0 to 0: 0: 0: 100 (v/v). Each elution solvent was analyzed by TLC, divided into 38 small fractions, concentrated under reduced pressure, and used as a first column chromatography fraction of hexane and ethyl acetate layers. Among the first column chromatography fractions, 10.85 g of the 23rd small fraction was used as a mobile phase using a mixed solvent of hexane and acetone (100: 0 ~ 1: 1, v/v), and 13 small fractions were prepared by medium pressure liquid chromatography. Obtained. Compound 1 (335 mg) was obtained from the fifth fraction (Fr.5) of these.

Hexane and ethyl acetate layer 2g of the 25th small fraction among the first column chromatography fractions was a mixed solvent of chloroform and methanol (100: 0 ~ 70: 30, v/v) as a mobile phase, using medium pressure liquid chromatography. A small fraction of dogs was obtained. Compound 2 (453.1 mg) was obtained from the fourth fraction (Fr.4) of these.

Hexane and ethyl acetate layer chromatography using a reverse phase filler (COSMOSIL 140C18-Prep.) with the seventh fraction (Fr.23-7) of 13 small fractions obtained from 10.85 g of the 23rd small fraction among the first column chromatography fractions. The graphical fraction was carried out. Through this, 6 small fractions were obtained, and compound 3 (157.4 mg) was obtained from the fourth fraction (Fr.23-7-4).

Example 2-2: Structural analysis of effective compounds

The molecular weights and molecular formulas of compounds 1, 2, and 3 obtained in Example 2-1 were determined using a liquid chromatography mass spectrometer (AGILENT1100). In addition, the molecular structure was determined using ¹ H NMR and ¹³ C NMR spectroscopy data through nuclear magnetic resonance (NMR) analysis (JEOL, JNM EX-400).

As a result of comparative analysis of the above instrumental analysis results with those of the published literature, ethyl gallate represented by the following formula (1), gallic acid represented by the following formula (2), aromadendrine represented by the following formula (3) (Aromadendrin) was confirmed. The detailed analysis results were as follows.

Compound 1: Ethyl gallate

[Formula 1]

1) Physical properties: white granular (m.p. 149-153 ℃)

2) Molecular weight: 198.17

3) Molecular formula: C ₉ H ₁₀ O ₅

4) 1H-NMR (methanol-d ₃ , 400 ㎒) δ 1.35(t), 4.27(dd), 7.04(s) 13C-NMR (methanol-d3, 100 ㎒) δ 14.78, 61.80, 110.17, 121.95, 139.84 , 146.64, 168.71

Compound 2: Gallic acid

[Formula 2]

1) Physical properties: White, yellowish-white crystals (m.p. 258-265 ℃)

2) Molecular weight: 170.12

3) Molecular Formula: C ₈ H ₆ O ₅

4) ¹ H-NMR (methanol-d ₃ , 400 ㎒) δ 7.05(s) ¹³ C-NMR (methanol-d ₃ , 100 ㎒) δ 110.48, 122.28, 139.69, 146.53, 170.60

Compound 3: Aromadendrin

[Formula 3]

1) Physical properties: White-gray, crystals (m.p. 228 ℃)

2) Molecular weight: 288.25

3) Molecular Formula: C ₁₅ H ₁₂ O ₆

4) ¹ H-NMR (methanol-d ₃ , 400 ㎒) δ 4.55(d), 5.88(d), 5.92(d), 6.83(d), 6.83(d), 7.35(d), 7.35(d) . ¹³ C-NMR (methanol-d ₃ , 100 ㎒) δ 73.6, 85.0, 96.3, 97.4, 101.8, 116.1, 116.1, 129.3, 130.4, 130.4, 159.2, 164.6, 165.3, 168.9, 198.5

Experimental Example 1: Confirmation of the effect of inhibiting the transcriptional activity of STAT3 induced by IL-6 in the extract, its fraction, and the first column chromatography fraction

The following experiment was performed to measure the effect of inhibiting the activity of STAT3 induced by IL-6 of the extract, its fraction, and the first column chromatography fraction.

Experimental Example 1-1: Preparation of transformant

PStat3-Luc, in which luciferase is expressed by STAT3 in Hep3B cells (ATCC HB-8064), and pcDNA3.1(+) (Clontech laboratories, Palo Alto, CA) were added to lipofectamin plus (Invitrogen, Carlsbad, CA, USA) Were transfected together. Two days after transfection, hygromycin was treated (100 µg/ml) to obtain a clone stably expressing luciferase. Whether luciferase is stably expressed in this clone was confirmed through a luciferase assay.

Experimental Example 1-2: IL-6 responsive STAT3 reporter gene test

The transfected cells were serum-free cultured with DMEM medium (GIBCO 119950965), and the samples were treated for 1 hour as follows, and then 10ng/ml IL-6 (R&D system, USA) was added and cultured for 12 hours. .

1: negative control group (non-treatment group);

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

3: Extract, fraction and hexane layer first column chromatography fraction (25, 50 μg/mL), ethyl acetate layer first column chromatography fraction (10, 30 μg/mL)

The reacted cells were washed with PBS, 50 µl lysis buffer (luciferase assay system, promega, USA) was added and stirred for 20 minutes, and then 30 µl to 100 µl of luciferase substrate (luciferase assay system, promega, USA) was added. The color development degree was measured within 5 minutes with a luminometer (EG&G BERTHOLD, USA), and the results are shown in FIGS. 1 and 2.

As a result, as shown in Figure 1, the ethanol extract of the present invention, and the hexane, ethyl acetate, and water fractions all showed a result of inhibiting the activity of STAT3 mediated by IL-6. Specifically, in the case of Ethanol extract, 83.9% and 101.0% at 25 ㎍ / ㎖ and 50 ㎍ / ㎖, respectively, and the hexane fraction of vinegar vine was 100.9% at 25 ㎍ / ㎖ and 50 ㎍ / ㎖, respectively. , 101.6% of inhibitory activity was shown, and the ethyl acetate fraction showed 89.1% and 101.1% inhibitory activities at 25 µg/ml and 50 µg/ml, respectively, and the water fraction 25 µg/ml and 50 µg/ml, respectively. Inhibitory activity was 19.4% and 99.2%.

In addition, in the case of the hexane layer primary column chromatography fraction, both fractions 1 to 10 showed a result of effectively inhibiting the activity of STAT3 at concentrations of 25 μg/ml and 50 μg/ml (FIG. 2), ethyl acetate layer 1 In the case of the secondary column chromatography fraction, the results were shown to effectively inhibit the activity of STAT3 at both 10 µg/ml and 30 µg/ml (Fig. 3). In the case of the isolated effective compounds, ethyl gallate and gallic acid exhibited 62.2% and 59.7% of STAT3 inhibitory activity, respectively, at a concentration of 60µg/ml, and aromadendrine showed lower STAT3 inhibitory activity than the two compounds. Shown (Fig. 4).

Experimental Example 1-3: IL-6-induced Stat3 and JAK2 phosphorylation inhibitory activity assay

After culturing Hep3B and U266 cells in a 6-well plate at 5×10 ⁵ cells/well at 80% confluency in a culture dish, exchanged for a serum-free medium and incubated for an additional 12 hours, and sampled as follows for 60 minutes. Processed.

1: negative control group (non-treatment group);

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

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

8: Genestein treatment group (100 μM)

Then, 20 ng/ml IL-6 was treated and reacted for 20 minutes, and then 40 µl lysis buffer (pH 8, 20 mM Tris-HCl, 137 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM Na3VO4, 2 mM EDTA) , 1mM PMSF, 20mM leupeptin, 20mg/ml aprotonin; Sigma, USA) to lyse the cells and centrifuge (13,000g, 15 minutes) to obtain a supernatant containing protein. Obtained. The concentration of the protein was quantified using a DC protein test kit (Bio-Rad, USA), and the protein was loaded on a 4-12% SDS polyacrylamide gel (SDS-PAGE), followed by electrophoresis at 175 mA for 2 hours. After electrophoresis was completed, the gel proteins were transferred to a PVDF membrane (Westran S, pore size 0.2 mm; Whatman, USA) at 35 V for 90 minutes. The transferred membrane was blocked with Tris-buffer solution (T-TBS; 50mM Tri-HCl, pH 7.6, 150mM NaCl, 0.2% Tween-20, 5% skim milk; Sigma, USA) for 1 hour at room temperature and then with T-TBS. Washed 5 times. The membrane was treated with polyclonal antibodies of phospho-JAK2 and phospho-Stat3 (1:1,000 dilution) as primary antibodies at 4° C. for 12 hours. After washing 5 times with T-TBS, HRP-binding anti-mouse antibody (1:5,000 dilution) was reacted with a secondary antibody for 1 hour. Then, it was washed with T-TBS and the film was developed using an ECL kit (Amersham, USA) in the dark. The results are shown in Figs. 5 and 6.

As a result, the ethanol extract of the present invention showed a result of effectively inhibiting the phosphorylation of STAT3 by IL-6 in a concentration-dependent manner (FIG. 5), and the phosphorylation of JAK2 (Tyr1007/1008) was also inhibited in a concentration-dependent manner. Is shown (Fig. 6).

The above results indicate that the ethanol extract of the present invention can effectively inhibit the signaling activity by IL-6. When viewed in combination with the results of Experimental Example 1-2, the extract of the present invention Alternatively, the use of a fraction thereof suggests that IL-6 mediated diseases can be effectively prevented or treated.

Experimental Example 2: Extracts and fractions of Amaranthus vines Osteoclast differentiation inhibiting effect induced by RANKL

Bone maintenance is regulated by osteoclasts absorbing old bones and osteoblasts consistently producing new bones. Therefore, an abnormal combination of osteoclasts and osteoblasts not only causes various bone diseases, but an increase in osteoclasts causes bone diseases such as osteoporosis, rheumatoid arthritis, and periodontitis. It is known that the activity of IL-6/STAT3 is importantly involved in the differentiation of osteoclasts induced by RANKL. Through Experimental Example 1, it was confirmed that the extract and fractions thereof of the present invention can effectively inhibit the activity of STAT3 induced by IL-6. Accordingly, an experiment was carried out as follows to determine whether the extract of vinegar vines has an inhibitory effect in the differentiation of osteoclasts induced by RANKL.

Experimental Example 2-1: RANKL-induced osteoclast differentiation inhibition efficacy test

Specifically, bone marrow cells were obtained by separating the thigh bone and shin bone of a 5-week-old mouse, and washing the inner space of the bone with a 1cc syringe. The isolated bone marrow cells were cultured for 3 days in a-MEM medium containing 10% FBS, antibiotics, and M-CSF (30 ng/ml). After 3 days, the attached cells were used as phagocytic cells (bone marrow macrophage, BMM). Phagocytic cells were cultured with the addition of M-CSF (30 ng/ml) and RANKL (50 ng/ml), and treated with extracts (50, 25 and 10 µg/ml) and fractions (50 µg/ml). After 4 days, the cultured cells were stained with a TRAP solution (Sigma Aldrich, USA), and the cells stained in red were judged as differentiated osteoclasts, and the results are shown in FIG. 7.

As a result, the ethanol extract of the present invention effectively inhibited the differentiation of osteoclasts induced by RANKL (Figs. 7A and B).

Experimental Example 2-2: Cytotoxicity test by XTT analysis

Phagocytic cells were added to a 96-well plate at a density of 1×10 ⁴ cells/well, and ethanol extract and M-CSF (30 ng/ml) were added and cultured for 3 days. After 3 days, 50 µl of the XTT solution was added to each well, and after incubation for 4 hours, the absorbance at 450 nm was confirmed using an ELISA reader (Molecular Devices, CA, USA), and the results are shown in FIG. 7C.

As a result, the Ethanol extract of the present invention did not show cytotoxicity at all, which suggests that the Ethanol extract has an effect of inhibiting the differentiation of osteoclasts irrespective of cytotoxicity (Fig. 7C).

Experimental Example 2-3: Expression analysis of genes / proteins that play an important role in osteoclast differentiation induced by RANKL

After RANKL binds to RANK in the cell membrane, it promotes the expression of transcription factors c-Fos and NFATc1 through intracellular signaling system and induces the expression of the TRAP gene, an indicator of osteoclasts. In RANKL stimulated BMM, mRNA expression of c-Fos, NFATc1, OSCAR, TRAP, cathepsin K, and DC-STAMP was quantified by real-time PCR analysis. After preparing BMM as in Experimental Example 2-1, ethanol extract (50 µg/ml) was pretreated for 1 hour, and RANKL (100 ng/ml) was added, followed by incubation for 12, 24, and 48 hours. After cultivation, RNA was isolated from each cell with a TRIzol (Invitrogen) solution, and cDNA was synthesized using 1 μg of the isolated RNA. The synthesized cDNA was amplified by PCR using the following primers.

gene order Sequence number 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 cathepsin K
sense 5'-CAGCAGAACGGAGGCATTGA-3' 9 antisense 5'-CCTTTGCCGTGGCGTTATAC-3' 10 DC-STAMP
sense 5'-GCAAGGAACCCAAGGAGTCG-3' 11 antisense 5'-CAGTTGGCCCAGAAAGAGGG-3' 12 GAPDH
sense 5'-ACCACAGTCCATGCCATCAC-3' 13 antisense 5'-TCCACCACCCTGTTGCTGTA-3' 14

As a result of PCR analysis, it was found that the ethanol extract of the present invention effectively inhibited the mRNA expression of all genes important for the differentiation of osteoclasts, c-Fos, NFATc1, OSCAR, TRAP, cathepsin K, and DC-STAMP ( Fig. 8). In particular, it could be seen that the ethanol extract of Asteraceae vine more effectively inhibited the expression of the transcription factor NFATc1 than the transcription factor c-Fos.

Experimental Example 2-4: Analysis of the activity of RANKL prior to subsignaling; Measurement of phosphorylation of p38, AKT , ERK , and I κB

After preparing BMM as in Experimental Example 2-1, it was replaced with a serum-free medium. After incubation in a serum-free medium for 3 hours, the ethanol extract of the vine was pretreated for 1 hour at each concentration. Thereafter, RANKL (100ng/ml) was added and stimulated for 15 minutes, and 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 blackberry vine inhibited the phosphorylation of p38 and IκB induced by RANKL, but did not affect the phosphorylation of AKT and ERK (FIG. 9).

Experimental Example 2-5: Bone resorption test

For the bone resorption test, osteoblasts were removed from the orbit (calvaria) of 1-day-old mice, and 0.1% collagenase and 0.2% dispase were added, and the extracted cells were mixed with 10% FBS and antibiotics ( penicillin/streptomycin) was obtained by incubating for 3 days in α-MEM culture solution. Bone marrow cells and osteoblasts were added to a 90-mm culture dish coated with collagen, treated with vitamin D ₃ (10 ^-8 M) and PGE2 (10 ^-6 M), followed by synchronous culture for 6 days. After 6 days, the attached mature 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 treated to remove osteoblasts, RANKL (100 ng/ml) was treated on pure osteoclasts, and ethanol extracts were treated with different concentrations for 24 hours. The osteoclasts were removed by treatment with 0.1% Triton X-100 and photographed with an optical microscope. The quantification of the bone resorption area is based on the Image Pro-plus program version, version. 4.0 (Media Cybernetics) was used.

As a result, it can be seen that the ethanol extract of the present invention effectively inhibits the bone resorption action of osteoclasts at a concentration of 50 µg/ml (FIG. 10).

In addition, in the present invention, it was confirmed that ethyl gallate, gallic acid, and aromadendrin, which are effective compounds isolated from black stalks through Experimental Example 1-3, exhibit excellent STAT3 inhibitory activity, whereby these compounds are induced by RANKL. It was examined whether there is an inhibitory effect in the differentiation of osteoclasts.

At this time, as in Experimental Example 2-1, the efficacy of inhibiting osteoclast differentiation induced by RANKL was examined.As a result, similar to the STAT3 inhibitory activity, ethyl gallate and gallic acid were excellent in osteoclast differentiation at concentrations of 10 μM and 30 μM. The inhibitory effect was shown, and aromadendrin was lower than that of the two compounds, but showed a result of effectively inhibiting the differentiation of osteoclasts at a concentration of 60 μM (FIG. 11).

Summarizing the above results, it was found that the extract, its fractions, and the compounds isolated therefrom of the present invention can treat IL-6-mediated diseases. This suggests that the disease can be effectively treated.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features thereof. In this regard, the embodiments described above are illustrative in all respects and should be understood as non-limiting. The scope of the present invention should be construed as including the meaning and scope of the claims to be described later rather than the above detailed description, and all changes or modifications derived from the equivalent concepts within the scope of the present invention.

<110> Korea Research Institute of Bioscience and Biotechnology <120> A pharmaceutical composition for preventing or treating IL-6-mediated disease, comprising extract, fraction, or compounds derived from Ampelopsis brevipedunculata <130> PA130871-KR-D1 <150> KR 10-2012-0112093 <151> 2012-10-09 <160> 14 <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 cathepsin K (sense) <400> 9 cagcagaacg gaggcattga 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for cathepsin K (antisense) <400> 10 cctttgccgt ggcgttatac 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for DC-STAMP (sense) <400> 11 gcaaggaacc caaggagtcg 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for DC-STAMP (antisense) <400> 12 cagttggccc agaaagaggg 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for GAPDH (sense) <400> 13 accacagtcc atgccatcac 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for GAPDH (antisense) <400> 14 tccaccaccc tgttgctgta 20

Claims (11)

A pharmaceutical for preventing or treating atopic dermatitis, which is an interleukin-6 mediated inflammatory disease, comprising an extract of Ampelopsis brevipedunculata stem or a fraction thereof including the compound represented by Formula 1 or 2 as an active ingredient Composition:
[Formula 1]

[Formula 2]

. The composition of claim 1, wherein the extract is obtained by extracting the buckthorn stem with a solvent selected from the group consisting of water, alcohols having 1 to 6 carbon atoms, and mixed solvents thereof. The composition of claim 2, wherein the alcohol is selected from the group consisting of ethanol, methanol and butanol. The composition of claim 1, wherein the fraction is obtained by dividing the extract of the wild vine stem with a solvent selected from the group consisting of hexane, ethyl acetate, water, and a mixed solvent thereof. The composition of claim 1, wherein the composition is an animal drug. Food for preventing or improving atopy, an IL-6 (interleukin-6) mediated inflammatory disease comprising an extract of Ampelopsis brevipedunculata stem or a fraction thereof including the compound represented by Formula 1 or 2 as an active ingredient Composition:
[Formula 1]

[Formula 2]

. A quasi-medicinal product for the prevention or improvement of atopic dermatitis, which is an IL-6-mediated inflammatory disease, comprising an extract of Ampelopsis brevipedunculata stem or a fraction thereof including the compound represented by Formula 1 or 2 as an active ingredient Composition:
[Formula 1]

[Formula 2]

. Feed for the prevention or improvement of atopic dermatitis, IL-6 (interleukin-6) -mediated inflammatory disease, comprising an extract of Ampelopsis brevipedunculata stem or a fraction thereof including the compound represented by Formula 1 or 2 as an active ingredient additive:
[Formula 1]

[Formula 2]

. Cosmetics for the prevention or improvement of atopic dermatitis, IL-6 (interleukin-6) -mediated inflammatory disease comprising an extract of the extract Ampelopsis brevipedunculata stem or a fraction thereof containing the compound represented by Formula 1 or 2 as an active ingredient Composition:
[Formula 1]

[Formula 2]

. A method of treating atopy, which is an IL-6 (interleukin-6) mediated inflammatory disease, comprising administering the composition of claim 1 to an individual except a human.
Drinking water for the prevention or improvement of atopy, an IL-6 (interleukin-6) mediated inflammatory disease, comprising an extract of Ampelopsis brevipedunculata stem or a fraction thereof including the compound represented by Formula 1 or 2 as an active ingredient additive:
[Formula 1]

[Formula 2]

.

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