KR101416149B1 - Composition comprising an extract of Curcuma longa L. or Curcuma aromatica L. isolated therefrom having IL-6 induced STAT3 inhibitory activity - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating IL-6-induced STAT3 mediated diseases comprising an over-the-ground extract of Curcuma longa L or Curcuma aromatica L, or a non-polar organic solvent fraction thereof as an active ingredient . Specifically, the present invention relates to a pharmaceutical composition for preventing or treating an IL-6-induced STAT3 mediated disease comprising as an active ingredient a turmeric or herbal topical extract or a nonpolar organic solvent fraction thereof, a pharmaceutical composition comprising the above pharmaceutical composition as an active ingredient A method of treating an IL-6-induced STAT3 mediated disease in an animal other than a human, comprising administering to the individual in need of treatment a functional food and a pharmaceutical composition for preventing or ameliorating an IL-6 induced STAT3 mediated disease . The topical extract of turmeric or coriander according to the present invention, its non-polar organic solvent fraction effectively inhibits IL-6 induced STAT3 activation, and thus can be effectively used for prevention and treatment of IL-6 induced STAT3 mediated diseases such as cancer or inflammatory diseases have.

Description

[0001] The present invention relates to a composition comprising a topical extract of turmeric or coriander having an inhibitory effect on IL-6-induced STAT3 activation, or a non-polar organic solvent fraction thereof, wherein the composition comprises an extract of Curcuma longa L. or Curcuma aromatica L. inhibitory activity}

The present invention relates to a pharmaceutical composition for preventing or treating IL-6-induced STAT3 mediated diseases comprising an over-the-ground extract of Curcuma longa L or Curcuma aromatica L, or a non-polar organic solvent fraction thereof as an active ingredient . Specifically, the present invention relates to a pharmaceutical composition for preventing or treating an IL-6-induced STAT3 mediated disease comprising as an active ingredient a turmeric or arachidaceous topical extract or a non-polar organic solvent fraction thereof, an IL- 6 induced STAT3 mediated disease in an animal other than a human, comprising administering to a subject in need of treatment a functional food for preventing or ameliorating an induced STAT3 mediated disease and the composition.

Interleukin-6 (IL-6) is a cytokine, also called B cell stimulating factor 2 (BSF2) or interferon beta2 (INF-beta2). IL-6 has been identified as a differentiator involved in the activation of B lymphocytes (Hirano, T. et al., Nature (1986) 324, 73-76). Thereafter, it was found to be a multifunctional cytokine that affects various cell functions (Akira, S. et al., Adv. In Immunology (1993) 54, 1-78). 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 a membrane-bound protein with a molecular weight of approximately 80 kDa expressed through the membrane. And the other is a membrane protein gp130 with 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 (Taga et al., J. Exp. Med. (1987) 166, 967). After binding of ligands and receptors, Janus Kinases 2 (JAK2) is activated in the cells 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. It is known that activated STAT3 binds to each other in the cytoplasm to form a homo- or hetero-dimer, enters the nucleus, binds to the recognition sequence of the target gene, and increases transcription (Levy, DE, et al., Nat Rev Mol Cell Biol, 2002, 3, 651-62, Darnell, JE, Jr., Science, 1997, 277, 1630-1635).

These IL-6-mediated signaling pathways have been reported to be associated with inflammatory diseases and various cancers, and thus inhibition of IL-6-mediated signaling pathways is therapeutically useful. Currently, anti IL-6 R antibodies are the most studied to study the inhibitory function of the signal transduction system of IL-6. This anti-IL-6R antibody has been reported to inhibit synovial cell growth against rheumatoid arthritis (International Patent Publication No. 98/11020), and is associated with proliferative hyperplasia, hyperimmunoglobulinemia, anemia, nephritis, cachexia, rheumatoid arthritis, (WO 96/12503), which is used for the treatment of diseases which contribute to IL-6 products such as cattle disease, cattle disease, and angina pectoris. It is also known in the prophylactic / protective agent for susceptible T-cell related diseases such as multiple sclerosis, uveitis, chronic thyroiditis, delayed hypersensitivity, contact dermatitis and atopic dermatitis (WO 98/42377) and used as a therapeutic agent for systemic erythromycin (International Patent Publication No. 98/42377). In addition, in a report used as a therapeutic agent for Crohn's disease (International Patent Publication No. 99/47170), its active ingredient is known as an anti-IL-6R antibody. Patents used as an active ingredient of a therapeutic agent for pancreatitis have also been reported (WO 00/10607), an anti-IL-6R antibody is also known as an active ingredient in International Patent Publication No. 02/3492, which is a patent for a therapeutic agent for psoriasis. In addition, an international anti- However, these proteins may have an epitope that can be recognized as an exogenous protein and may still be immunogenic when used as a therapeutic agent. However, in the case of protein Many small molecule compounds are not recognized by the immune system.

In addition, the IL-6 signaling pathway associated with cancer is much related to its intermediate mediator, STAT3. It has been reported to be involved in a variety of cancers including myeloma, breast, prostate, brain, 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 cancers have been found to have structurally activated STAT3, STAT3 being associated with some acute leukemias (Gouilleux-Gruart, V. et al., Leuk. Lymphoma, 1997, 28, 83-88) and T Have been found to be structurally active in cellular lymphomas (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, DA, et al., J. Clin. Invest., 1997, 100, 3140-3148). STAT3 has been found to be structurally active in myeloma tumor cells, both in bone marrow mononuclear cells and in cultures from patients with multiple myeloma. These cells are resistant to Fas-mediated cell death 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). In recent years, IL-6 is secreted and IL-6 is eliminated ideally in cancer patients induced by Ras, including pancreatic cancer. In addition, tumor growth and angiogenesis caused by Ras are inhibited and tumor size is reduced (Brooke Ancrile et al., Gene & Development, 2007, 21, 1714-1719). In addition, IL-6-induced gp130 / JAK / STAT3 pathway is expected to be a new target for chemotherapy, as IL-6 is overexpressed in EGFR-mutated lung adenocarcinoma and STAT3 is activated (Sizhi Paul Gao et al., J. Clin. Invest. 2007, 117, 38463856).

Meanwhile, Curcuma Longa L and Curcuma aromatica L are the annual plants of ginger and Zingiberaceae, native to tropical Asia, cultivated in various areas except southern China and the mountainous regions of northern China, The components include turmerone, zingerene, phellandrene, 1,8-cineole, borneol, dehydroturmerone and the like, , P-hydroxy cinnamoyl feruloyl methane, which is a derivative of the diketone compound curcumin and derivatives thereof, which contains a fructose, glucose, starch, organic acid, And p, p'-dihydroxy dicinnamoyl methane (0.3%). In addition, it contains 1 ~ 5% of essential oil, 2.4% of nonvolatile oil, 50% of starch, , Crude fiber 5%, ash 4%, moisture 16% All. It is known that it plays a role in promoting blood circulation and is used as a treatment for bruise or hemorrhoids including shoulder joint pain, and has a suppressive effect on blood cholesterol lowering action and hepatic viral infection In particular, curcumin is known to be effective for the treatment of dental carcinoma and anti-inflammatory disease (Korean Patent No. 10 / (Bharat B. Aggarwal et al., Phytopharmaceuticals in Cancer Chemoprevention, 2004) have been known to be used for inhibiting angiogenesis, inducing apoptosis, inhibiting metastasis, decreasing cholesterol, immunosuppressing effect, and HIV therapeutic effect.

In the present invention, curcumin, demethoxy curcumin and bisdemethoxy curcumin were not detected in the stem part (ground part) of turmeric or coriander (Korea Patent Publication No. 2011/0004763). However, Extract or fraction thereof inhibits IL-6-induced STAT3 activation and inhibits the IL-6 signal transduction system. Thus, even though it has been used as a food for a long time, its base Has been used for the treatment of cancerous or inflammatory diseases, and thus the present invention has been completed.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating IL-6-induced STAT3 mediated diseases, which comprises an extract of turmeric or coriander, or a non-polar organic solvent fraction thereof as an active ingredient.

Another object of the present invention is to provide a functional food for prevention or improvement of an IL-6-induced STAT3-mediated disease comprising an extract of a turmeric or corn ground portion, or a non-polar organic solvent fraction thereof.

Yet another object of the present invention is to provide a method of treating an IL-6-induced STAT3 mediated disease in an animal other than a human, comprising administering the pharmaceutical composition to a subject in need of treatment.

In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating an IL-6-induced STAT3 mediated disease comprising an extract of turmeric or corn groundworts, or a non-polar organic solvent fraction thereof as an active ingredient.

The turmeric or lupine extracts contained in the pharmaceutical composition of the present invention include any of the extracts, soluble extracts, diluted solutions or concentrates obtained in the respective steps of the extraction treatment, or the dried products thereof. Preferably, the methanolic cold extract of turmeric or coriander is used, more preferably the ethyl acetate-soluble extract of methanolic cold extract is used.

In addition, the nonpolar organic solvent fraction contained in the composition includes any of the fractions and the purified product obtained in each step of the purification treatment using the extract, the diluted solution thereof, or the concentrated solution or the dried product thereof. Preferably, the hexane: acetone fraction of the methanolic cold extract of the turmeric or corn steep part is used.

The topical extract of turmeric or coriander having the IL-6-induced STAT3 activation inhibitory effect from the above-ground part of turmeric or coriander according to the present invention can be produced by the following method.

Specifically, the method for producing a topical extract of turmeric or coriander of the present invention comprises extracting a top portion of turmeric or coriander with water, an organic solvent or a mixed solvent thereof.

Preferably, the ground part of turmeric or coriander ground by drying for a certain period of time can be extracted by various extraction methods such as cold extraction, heat extraction, ultrasonic extraction, and reflux cooling extraction as known in the art. The extraction method is not particularly limited and may be carried out at room temperature or by heating under conditions where the active ingredient is not destroyed or its destruction is minimized.

The above-ground parts of the turmeric or coriander can be used without restrictions such as cultivated or marketed, cleaned and dried. Examples of the organic solvent suitable for the present invention include methanol, ethanol, isopropanol, butanol, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, dichloromethane, N, N- (C ₁ ) to 4 (C ₄ ) lower alcohols such as methanol, ethanol and the like, and more preferred are alcohols such as methanol, ethanol, Can be extracted using methanol.

In a preferred embodiment of the present invention, the top portion of turmeric or coriander is washed and shredded, pulverized and pulverized and then pulverized to 2 to 20 times, preferably 3 to 5 times the weight of the top portion of dried turmeric or coriander Extraction with water, organic solvent or mixed solvent thereof at a temperature of about 15 to 100 ° C for about 12 hours to 4 days using a method such as hot water extraction, cold extraction, reflux cooling extraction or ultrasonic extraction, The liquid is recovered. This extraction process can be repeated several times, preferably repeated twice, collecting the supernatant, and concentrating it under reduced pressure or lyophilizing to obtain a topical extract of turmeric or turmeric.

In addition, the non-polar organic solvent fraction having a high activity from the over-the-ground extract of turmeric or coriander contained in the composition of the present invention is further separated by a method such as chromatography to thereby inhibit IL-6 induced STAT3 activation inhibition according to the present invention The turmeric or urea fractions can be separated.

Specifically, a method for purifying a turmeric or ganoderma fractions from the above-ground portion of turmeric or ganoderma according to the present invention comprises the steps of: 1) fractionally extracting the above-ground Ganoderma lucidum or Ganoderma lucidum extract with a nonpolar organic solvent to obtain a non-polar organic solvent soluble extract; And 3) purifying the obtained non-polar organic solvent-soluble extract by chromatography.

Step 1) is a step of suspending the topical extract of turmeric or coriander with water and sequentially extracting it with a nonpolar organic solvent to obtain a nonpolar organic solvent-soluble extract. At this time, a usual fractionation extraction method can be used, and a fractionation funnel can be preferably used. The nonpolar organic solvent suitable for the present invention is hexane, ether, dichloromethane, chloroform, ethyl acetate or a mixed solvent thereof, preferably fractionated using ethyl acetate.

Step 2) is a step of purifying the above-obtained non-polar organic solvent soluble extract of turmeric or coriander obtained by chromatography to separate the turmeric or urea fractions. In the present invention, the active ingredient can be isolated by performing chromatography on the overhead nonpolar organic solvent-soluble extract of turmeric or coriander, and the kind of the chromatography column and the developing solvent can be variously controlled.

According to an embodiment of the present invention, 3 kg of ethanol was added to 1 kg of pulverized turmeric or coriander, and the mixture was cooled and extracted at room temperature for 3 days. After filtration, the filtrate was concentrated under reduced pressure to obtain 60 g of a turmeric or turmeric crude extract. The extract was fractionated with ethyl acetate to obtain 12.73 g of nonpolar organic solvent soluble extract. This was concentrated under reduced pressure, and then subjected to silica gel column chromatography (hexane: acetone = 100/0 to 0/100 gradient) to obtain 900 mg of a fraction.

Additionally, the pharmaceutical composition of the present invention may comprise a pharmaceutically effective amount of a topical extract of turmeric or corn, its non-polar organic solvent fraction, or may comprise one or more pharmaceutically acceptable carriers, excipients or diluents have.

The term "IL-6 induced STAT3 mediated disease" in the present invention refers to a disease mediated by excessive activation of STAT3, which is known to be associated with excessive activation of IL-6 and thus cancer induced, do.

In the present invention, the term "cancer" refers to a disorder in the function of regulating normal division, differentiation and death of cells, resulting in abnormally proliferative hyperproliferation and invasion into surrounding tissues and organs to form lumps, Such as pancreatic cancer, breast cancer, prostate cancer, brain tumor, head and neck carcinoma, melanoma, myeloma, leukemia, lymphoma, liver cancer, gastric cancer, colon cancer, bone cancer, uterine cancer, ovarian cancer, rectal cancer, , Colon cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, bladder cancer, renal cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma and central nervous system tumor. It does not.

In the present invention, the term "inflammation " refers to a defense reaction of biological tissue against a certain stimulus, and refers to a complicated lesion involving tissue degeneration, circulatory disorder and exudate, and tissue proliferation. In addition, various types of infection and expression in the in vivo defense system against irritants in metabolites in vivo, various chemical mediators are involved in the mechanism of expression of inflammation, and the pathogenesis thereof is very complicated. It is a topical protective response caused by tissue injury or destruction, which acts to destroy, weaken or mask both injurious and injurious tissues. The characteristic of this inflammation is that the microvessels are perforated, the blood components leak into the interstitial space, and the white blood cells migrate to the inflammatory tissues, usually accompanied by clinical symptoms such as erythema, edema, hyperalgesia and pain. Examples of such diseases related to inflammation include rheumatoid arthritis, osteoporosis, transglottial hyperplasia, hyperimmunoglobulinemia, anemia, nephritis, cachexia, cattle disease, angioplasty nephritis, multiple sclerosis, uveitis, chronic thyroiditis, But are not limited to, dermatitis, systemic erythematosus, Crohn's disease, pancreatitis, psoriasis, burning idiopathic atrophy, diabetes and Alzheimer's.

The term "prophylactic" in the present invention refers to the administration of a pharmaceutical composition for prevention or treatment of an IL-6 induced STAT3 mediated disease comprising as an active ingredient a topical extract of turmeric or coriander according to the present invention, its non-polar organic solvent fraction, 6 < / RTI > induced STAT3 mediated disease.

The term "treatment" in the present invention refers to the administration of a pharmaceutical composition for prevention or treatment of an IL-6-induced STAT3 mediated disease comprising as an active ingredient a topical extract of turmeric or coriander according to the present invention, 6 Induction All actions that improve or improve the symptoms of STAT3-mediated disease.

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 depend on the patient's sex, age, 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 pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with another therapeutic agent, and may be administered sequentially or simultaneously with a conventional therapeutic agent. In addition, the pharmaceutical composition of the present invention 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. Specifically, the pharmaceutical composition of the present invention is preferably administered orally or intravenously.

Examples of pharmaceutically acceptable carriers, excipients and diluents that can be used in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, , Gelatin, calcium phosphate, calcium silicate, calcium carbonate, cellulose, methylcellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like.

The pharmaceutical composition of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method . In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like which are generally used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin And the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances and preservatives are included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

In a preferred embodiment of the present invention, the present invention provides a method for inhibiting the expression of IL-6-induced protein (Fig. 1 and Fig. 2) and IL-6 induced STAT3 (FIG. 3) and inhibited the migration of phosphorylated STAT3 into the nucleus (FIG. 4). In addition, it was confirmed that STAT3-induced expression of SOCS-3 was also inhibited.

Therefore, the pharmaceutical composition according to the present invention contains an extract of turmeric or urea having the effect of inhibiting IL-6 induced STAT3 activation, and its non-polar organic solvent fraction as an active ingredient, and is useful for prevention or treatment of IL-6 induced STAT3 mediated diseases It is expected that it can be used as well as preventing or treating the symptom or complication caused thereby.

In another aspect, the invention is directed to a method of treating an IL-6 induced STAT3 mediated disease in an animal other than a human, comprising administering the pharmaceutical composition to a subject in need thereof.

The method of treatment according to the present invention does not mean that a method of treating an animal other than a human, but such a treatment method is ineffective in humans. In addition, in consideration of having a disease in which symptoms can be improved by administering a pharmaceutical composition according to the present invention that inhibits IL-6 induced STAT3 activation in cells in a human case, it is sufficiently used in human therapy Can be.

The term "an animal other than a human being" in the present invention refers to an animal, excluding humans having a disease whose symptoms can be alleviated by the administration of the pharmaceutical composition according to the present invention which inhibits IL-6 induced STAT3 activation in cells, Pig, goat, camel, nutrition, and dog. Induced STAT3 mediated diseases, such as cancer, by administering to a mammal a pharmaceutical composition comprising, as an active ingredient, a topical extract of turmeric or lupine having an inhibitory effect on IL-6 induced STAT3 activation, and a nonpolar organic solvent fraction thereof, And inflammatory diseases can be effectively prevented and treated.

The term "administering" as used herein refers to the introduction of a predetermined substance into an animal by any suitable method, and the route of administration of the pharmaceutical composition according to the present invention may be administered orally, via any ordinary route, May be administered parenterally. In addition, the pharmaceutical composition according to the present invention may be administered by any device capable of moving the active ingredient into the target cell.

The preferred dosage of the pharmaceutical composition according to the present invention varies depending on the condition and the weight of the patient, the degree of the disease, the drug form, the administration route and the period, but can be appropriately selected by those skilled in the art. However, for the desired effect, the pharmaceutical composition of the present invention is preferably administered at a dose of 50 to 1000 mg / kg, preferably 100 to 500 mg / kg per day, and the linolanol compound is administered at a dose of 1 to 10 mg per day / kg, preferably 1 to 5 mg / kg. The administration may be carried out once a day or divided into several times.

In another aspect, the present invention provides a functional food for preventing or ameliorating an IL-6 induced STAT3 mediated disease, comprising an extract of turmeric or coriander, or a non-polar organic solvent fraction thereof.

Specifically, the topical extract of turmeric or coriander according to the present invention, the non-polar organic solvent fraction thereof, can be added to food or beverage for the purpose of improving or preventing IL-6 induced STAT3 mediated diseases. The type of food is not particularly limited For example, various foods such as confectionery, bakery products and noodles, drinks such as water, soft drinks and fruit drinks, gum, tea, vitamin complex, seasoning, and health functional foods. At this time, the amount of the extract or the fraction in the food or beverage may be generally 0.01 to 15% by weight, preferably 0.1 to 5% by weight of the total food weight in the case of the health functional food composition of the present invention, May be added in a proportion of 0.01 to 5.0 g, preferably 0.01 to 1.0 g, based on 100 ml. Since the health functional food of the present invention thus obtained contains the composition of the present invention, it is a food which can fully utilize the health prevention and therapeutic effects of the extract or fraction according to the present invention.

The health beverage composition of the present invention is not particularly limited to a liquid ingredient other than the above-mentioned extract or fraction as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages . Examples of the above-mentioned natural carbohydrate include monosaccharides such as glucose and fructose: disaccharides such as maltose, sucrose and the like; Polysaccharides such as dextrin, cyclodextrin and the like; And sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavorings (taumakin, stevia extract, etc.) and synthetic flavorings (saccharin, aspartan, etc.) can be advantageously used as flavorings other than those described above. The ratio of the natural carbohydrate is generally about 0.1 mg to 2.0 g, preferably about 0.1 mg to 1.0 g per 100 mL of the composition of the present invention.

 The health functional food of the present invention can be obtained by adding the above-described extract or compound of the present invention to a foodstuff of a base material or by adding the above-mentioned extract or fraction of the present invention after preparation of a foodstuff to be a base And then adding it. At this time, a taste and odor corrector may be added as needed.

In addition to the above, the health functional food of the present invention may contain flavorings such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and heavies (cheese, chocolate etc.), pectic acid and its salts, And salts thereof, organic acids, protective colloid concentrating agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the health functional food of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. Such components may be used independently or in combination. The proportion of such additives is generally selected within a range of about 20 parts by weight or less per 100 parts by weight of the health functional food of the present invention.

The topical extract of turmeric or coriander according to the present invention, its non-polar organic solvent fraction effectively inhibits IL-6 induced STAT3 activation, and thus can be effectively used for prevention and treatment of IL-6 induced STAT3 mediated diseases such as cancer or inflammatory diseases have.

FIG. 1 is a graph showing the inhibitory activity of IL-6-induced luciferase expression of the methanol extract and ethyl acetate fraction of the ground wart.
2 is a graph showing the inhibitory activity of IL-6-induced luciferase expression of the ethyl acetate fraction of the turmeric top portion.
FIG. 3 is a graph showing the IL-6-induced STAT3 phosphorylation inhibitory activity of the ethyl acetate fraction of the turmeric top part.
FIG. 4 is a photograph showing the STAT3 nuclear transfer inhibition effect of IL-6-phosphorylated ethyl acetate fraction of the turmeric surface part.
FIG. 5 is a graph showing STAT3-induced SOCS-3 expression inhibiting activity of the ethyl acetate fraction of the turmeric top part.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1 Extraction and Purification of Active Substance Having IL-6 Induced STAT3 Activation Inhibiting Activity from Turmeric or Wiltwood

 Turmeric or coriander (leaves and stems) were thoroughly washed with water, dried in the shade, and powdered with a Waring brand. Each of the powdered samples was added to methanol, followed by cold extraction at room temperature for 3 days, followed by filtration under reduced pressure through a filter paper (Wattmann, USA). The filtrate was filtered through a vacuum rotary evaporator at room temperature to remove the methanol solvent, 60 g of ground trout extract of Ulugum were obtained, respectively.

In order to separate and purify the active substance from the crude extract, the crude sulfur extract was suspended in 1 L of water and then mixed with the same amount of ethyl acetate. The reaction was repeated 4 times to obtain 1 L of water-soluble fraction, After obtaining 4 L of a fraction, the ethyl acetate soluble fraction was concentrated under reduced pressure to obtain 12.73 g of an ethyl acetate soluble extract.

12.73 g of the ethyl acetate soluble extract obtained above was purified by column chromatography on silica gel using a step gradient solvent system consisting of hexane: acetone = (100/0 to 0/100, v / v) The active fractions were separated and 900 mg of the ethyl acetate fraction was obtained.

< Example  2> curcuma  or Ulgum District Cucuminoid system  Investigation of compound content

The concentrations of curcumin, demethoxy curcumin and bisdemethoxy curcumin were adjusted to 15.625, 31.25, 62.5, 125, 250, 500, and 500 mg, respectively, in order to confirm the presence of the glucuronide- 1000 占 퐂 / ml, and analyzed by the following conditions to obtain a calibration curve.

The HPLC analysis used in this example used the company's pump system with an Agilent 1200 series HPLC instrument. The detector used in this example was tested at 260 nm, the best analytical detection wavelength using a VWD (variable wavelength detector), and the solvent flow rate was 1.0 ml / min. The sample injection volume was 10 μl, and the concentration was 10 mg / ml. Columns of the HPLC analysis were analyzed using ZORBAX-SB-18 (5 [mu] m, 150 mm x 4.6 mm). The mobile phase analysis was carried out with 20% acetonitrile, 0 min, with polar partitioning conditions of water (containing 0.1% TFA) and acetonitrile (containing 0.1% TFA). 25% acetonitrile, 10 min; 35% acetonitrile, 20 min; 50% acetonitrile, 30 min; 60% acetonitrile, 40 min; 70% acetonitrile, 50 min; 100% acetonitrile, 60 min.

The contents of curcumin, demethoxy curcumin and bisdemethoxy curcumin in methanol extracts of turmeric or turmeric were determined on the basis of the above-mentioned calibration curve.

Used sample Content (g / kg, extract) Curcumin Demethoxycoumine Bisdemethoxycoumine Root roots ethanol extract 17.0 5.3 3.4 Stem ethanol extract - - - Rootstooth ethanol extract 2.8 0.4 6.8

As shown in Table 1, 17.0 g / kg of curcumin (extract), 5.3 g / kg of demethoxy curcumin (extract) and 3.4 g / kg of bisdemethoxy curcumin (extract) were detected at the roots of the root, The contents of curcumin 2.8 g / kg (fraction), demethoxy curcumin 0.4 g / kg (fraction) and bisdemethoxy curcumin 6.8 g / kg (fraction). However, in the stem region, curcumin, demethoxy curcumin and bisdemethoxy curcumin Was not detected.

Similar results were also obtained in the extracts of each part of turmeric.

These results indicate that curcumin and its derivatives are not present in the topical extract of turmeric or corn, and that the efficacy of the extracts and fractions according to the present invention is caused by an active substance different from curcumin.

< Example  3> The extract of the present invention and The fraction IL -6 Induction study

<3-1> Ulgum  Ground-top extract and Fraction IL -6 induction Luciferase  Inhibition activity assay

In order to investigate the effect of the topical extracts and fractions of Ulvae on the expression mechanism of IL-6-related proteins, the following experiment was conducted.

HepG2 cells (ATCC HB-8065) were dispensed into a 96-well plate at 5 × 10 ⁴ cells / well and then treated with 10% FBS (v / v), 60.0 mg / l kanamycin sulfate (Gibco. The cells were cultured in a DMEM culture medium containing 5 g / l sodium bicarbonate (NaHCO ₃ ; Sigma, USA) at 80% confluent in a culture dish under the condition of 5% CO ₂ at 37 ° C. Subsequently, the medium was replaced with 50 μl of serum-free medium, and a mixture of 0.1 μg pSTAT3-TA-Luc (Clontech, CA) and 0.3 μl lipofectamine reagent (Invitrogen, USA) was added to each well. -TA-Luc was transfected and replaced with freshly prepared 200 [mu] l DMEM culture medium and incubated for an additional 24 hours.

The transfected cells were serum-starvated with 1% BSA / DMEM, treated with 1 ng / ml IL-6 (R & D system, USA) for 3 hours .

1: negative control group (untreated group);

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

3: Extracts and fractions (10, 30 μg / mL); And

4: Genistein (60 [mu] M);

The cells were washed with PBS, and 50 μl lysis buffer (luciferase assay system, promega, USA) was added. After stirring for 1 minute, 30 ~ 100 μl of luciferase assay system (promega, USA) The degree of color development was measured with a luminometer (EG & G BERTHOLD, USA) within 5 minutes and shown in FIG.

FIG. 1 is a graph showing the inhibitory activity of IL-6-induced luciferase expression of the methanol extract and ethyl acetate fraction of the ground wart. As shown in Fig. 1, both the methanol extract and the ethyl acetate fraction of Ulmus showed luciferase activity of less than 0.013, which was lower than the activity of 0.023 or more of the positive control treated with IL-6 alone.

These results indicate that the methanol extract and ethylacetate fractions of E. coli have the effect of inhibiting the expression of STAT3 luciferase induced by IL-6.

<3-2> Turbulent  Ground-top extract and Fraction IL -6 induction Luciferase  Inhibition activity assay

In order to investigate the effect of the topical extracts and fractions of turmeric on the expression mechanism of IL-6-related proteins, the following experiment was conducted.

Hep3B cells (ATCC HB-8064) were transfected together with pStat3-Luc and pcDNA3.1 (+) (Clontech laboratories, Palo Alto, Calif.) With lipofectamine plus (Invitrogen, Carlsbad, . Two days later, hygromycin was treated (100 占 퐂 / mL) to obtain a clone in which luciferase was stably expressed. Whether luciferase was stably expressed in this clone was confirmed by luciferase activity assay.

The transfected cells were serum-starvated with DMEM (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 (IL-6 10 ng / mL);

3: fractions (10, 30, 60 [mu] g / mL);

After washing the cells with PBS, 50 μL lysis buffer (luciferase assay system, promega, USA) was added and stirred for 20 minutes. 30 ~ 100 μL of luciferase substrate was added and the color development degree was measured with a luminometer The results are shown in Fig.

FIG. 2 is a graph showing the inhibitory activity of IL-6-induced luciferase expression of the ethyl acetate fraction of the turmeric top part. As shown in FIG. 2, the ethyl acetate fraction of turmeric was similar to that of the positive control group treated with IL-6 only at a concentration of 10 μg / mL, but the activity was inhibited as the concentration increased, resulting in a concentration of 60 μg / Activity of about 0.1 was observed.

These results indicate that the ethylacetate fraction of turmeric has the effect of inhibiting the expression of STAT3 luciferase induced by IL-6 in a concentration-dependent manner.

< Example  4> The extract of the present invention and The fraction IL -6 induction STAT3  Influence on phosphorylation

In order to investigate the effect of the extract and fraction of the present invention on the phosphorylation, a mechanism of regulating the activity of IL-6-induced STAT3 protein, the following experiment was conducted.

Hep3B cells were seeded at 6 × 10 ⁵ cells / well in a 6-well plate and cultured in a culture dish to a full 80%. Then, the medium was replaced with serum-free medium and cultured for another 12 hours.

1: negative control group (untreated group);

2: positive control (IL-16 10 ng / mL);

3: Fractions (10, 30, 60 [mu] g / ml)

8: Genestein treated group (60 [mu] M).

After 20 ng / mL IL-6 by processing after reaction for 20 minutes 40 μL lysis buffer (pH 8, 20 mM Tris- HCl, 137 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM Na 3 _{VO 4, 2 mM EDTA, 1} mM PMSF, 20 mM leupeptin (leupeptin), 20 mg / mL O picture Nin (aprotonin); 15 bungan centrifugation, 13000 g was dissolved in the cell using the Sigma, USA) To obtain a supernatant in which the protein was dissolved. Protein concentration was quantified using a DC protein test kit (Bio-Rad, USA) and the proteins were loaded onto 4-12% SDS polyacrylamide gel (SDS-PAGE) and electrophoresed at 175 mA for 2 h. 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 Tris-buffer (T-TBS; 50 mM Tri-HCl, pH 7.6, 150 mM NaCl, 0.2% Tween-20, 5% skim milk; Sigma, USA) And washed 5 times with T-TBS. The membrane was treated with phospho-STAT3 (1: 1000 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: 5000 dilution) was reacted with secondary antibody for 1 hour. After washing with T-TBS, the film was developed using an ECL kit (Amersham, USA) in a dark room. The results are shown in FIG. 3 as a result of the ethyl acetate fraction of the turmeric surface part.

FIG. 3 is a graph showing the IL-6-induced STAT3 phosphorylation inhibitory activity of the ethyl acetate fraction of the turmeric top part. As shown in FIG. 3, when compared with the amount of the same STAT3 protein, the ethylacetate fraction of turmeric induced phosphorylation similar to that of the positive control treated with IL-6 at a concentration of 10 μg / mL, . In addition, it was confirmed that the extracts and fractions of UGT also inhibited phosphorylation of STAT3 in a concentration-dependent manner (no result).

These results indicate that the ethylacetate fraction of turmeric inhibits the activity of STAT3 by inhibiting IL-6-induced phosphorylation of STAT3. Indicating that it has the effect of inhibiting the sub-mechanisms mediated by the activity of STAT3 in a concentration-dependent manner.

< Example  5> The extract of the present invention and The fraction IL -6 induced phosphorylation STAT3 of Nucleus  Investigation on the movement

In order to investigate the effect of the extract and fraction of the present invention on migration into the nucleus, which is an intracellular acting site of IL-6-induced phosphorylated STAT3 protein, experiments were conducted as follows.

Hep3B cells were plated at 8 × 10 ⁴ cells / well in an 8-well plate and cultured in a culture dish to a full 80%, then exchanged with a serum-free medium and further cultured for 12 hours, and the sample was treated for 60 minutes as described below.

1: negative control group (untreated group);

2: positive control (IL-16 10 ng / mL);

3: Fractions (30 μg / mL) and

4: Genestein treated group (60 [mu] M).

Subsequently, the cells were treated with 10 ng / mL IL-6 and reacted for 2 hours. The supernatant was removed and washed three times with PBS. Cells were fixed with 200 μl of 4% paraformaldehyde, After washing twice, the cells were permeabilized with 100% methanol. The cells were blocked with 1% BSA and reacted with STAT3 (1: 200 dilution) as a primary antibody overnight at 4 ° C. The cells were washed three times with PBS for 5 minutes each and then incubated with FITC-conjugated anti- 1: 1000 dilution) for 1 hour. After washing three times with PBS for 5 minutes each, fixation was carried out on a slide using a slowfade gold antifade reagent, and the change in nuclear distribution of STAT3 was observed through a confocal microscope (carl zeiss LSM 510 META confocal microscope) As a result, the results of the fraction of ethyl acetate fraction of the turmeric surface part are shown in FIG.

FIG. 4 is a photograph showing the STAT3 nuclear transfer inhibition effect of IL-6-phosphorylated ethyl acetate fraction of the turmeric surface part. As shown in FIG. 4, when the concentration of the ethyl acetate fraction of the turmeric was 30 μg / mL, STAT3 was concentrated in the nucleus in the IL-6-treated control group, whereas the fraction of turmeric ethyl acetate In the treated group, similar to the negative control group, they were found to be uniformly distributed in the cytoplasm. In addition, similar results were obtained when treating the extracts and fractions of Ulvae (results not yet obtained).

These results indicate that the ethylacetate fraction of turmeric inhibits the expression of genes expressed by activation of STAT3 by inhibiting the migration of IL-6-induced phosphorylated STAT3 into the nucleus. Indicating that it has the effect of inhibiting the sub-mechanisms mediated by the activity of STAT3.

< Example  6> The extract of the present invention and The fraction STAT3  Induced SOCS -3 expression

In order to investigate the effect of the extract and fraction of the present invention on the expression of SOCS-3, which is known to be induced by activation of STAT3 protein, the following experiment was conducted.

Hep3B cells were seeded at a density of 5 × 10 ⁴ cells / well in a 6-well plate and cultured to a full 80% in a culture dish. Then, the medium was replaced with serum-free medium and cultured for another 12 hours.

1: negative control group (untreated group);

2: positive control (IL-16 10 ng / mL);

3: Fractions (10, 30, 60 [mu] g / ml)

8: Genestein treated group (60 [mu] M).

Then, the cells were treated with 20 ng / mL IL-6 and reacted for 6 hours. The cells were collected in tubes and centrifuged to remove the medium. After washing with PBS, the cells were washed with RNeasy Mini Elute RNA was extracted using a Cleanup kit. The concentration and purity of the RNA were measured with a 2100 Bioanalyzer system (Agilent Technologies) and cDNA was synthesized using Maxime RT PreMix (Random primer, iNtRON Biotechnology, INC).

The degree of expression of SOCS-3 was measured by real-time PCR using a Taqman PCR master mix kit (Applied Biosystem), and the results of the fraction of the turmeric topoisomeric ethyl acetate were shown in FIG. 5 Respectively.

FIG. 5 is a graph showing STAT3-induced SOCS-3 expression inhibiting activity of the ethyl acetate fraction of the turmeric top part. As shown in FIG. 5, when the ethylacetate fractions of turmeric were treated at the concentrations of 10, 30 and 60 μg / mL, the expression levels of about 50, 30 and 10, respectively, were 90 or more of the positive control group treated with IL-6 And the expression level was low in comparison with the expression level. In addition, similar results were obtained when treating the extracts and fractions of Ulvae (results not yet obtained).

These results indicate that the ethyl acetate fraction of turmeric has the effect of inhibiting the expression of SOCS-3 induced by phosphorylated STAT3.

According to the results of the above examples, the present invention relates to an extract of corn or oriental herb extract and a non-polar organic solvent fraction thereof, which can be used to treat cancer and rheumatoid arthritis caused by excessive activity of IL-6 induced STAT3, osteoporosis, Aplastic anemia, atopic dermatitis, systemic erythematosus, Crohn's disease, pancreatitis, psoriasis, inflammatory idiopathic diabetes mellitus, diabetic retinopathy, diabetic retinopathy, diabetic retinopathy, diabetic retinopathy, It can be effectively used for the prevention and treatment of inflammatory diseases such as Alzheimer's disease.

Examples of formulations for the composition of the present invention are illustrated below.

&Lt; Formulation Example 1 > Preparation of pharmaceutical preparation

The pharmaceutical preparations containing the topical extract of turmeric or coriander according to the present invention and the nonpolar organic solvent fraction thereof as an active ingredient were prepared as follows.

<1-1> Preparation of powder

Topical extract of turmeric or turmeric, its nonpolar organic solvent fraction 2 g

Lactose 1 g

After mixing the above components, they were packed in airtight bags to prepare powders.

<1-2> Preparation of tablets

Topical extract of turmeric or turmeric, its nonpolar organic solvent fraction 100 mg

Corn starch 100 mg

Lactose 100 mg

2 mg of magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional tablet preparation method.

&Lt; 1-3 > Preparation of capsules

Topical extract of turmeric or turmeric, its nonpolar organic solvent fraction 100 mg

Corn starch 100 mg

Lactose 100 mg

2 mg of magnesium stearate

After mixing the above components, the capsules were filled in gelatin capsules according to a conventional capsule preparation method.

<1-4> Preparation of Injection Solution

Topical extract of turmeric or coriander, its nonpolar organic solvent fraction 10 ㎍ / ml

Until dilute hydrochloric acid BP pH 3.5

Sodium chloride BP injected up to 1 ml

The appropriate amount of the high-water seed extract according to the present invention, the non-polar organic solvent fraction thereof or the linerol compound separated therefrom is dissolved in sodium chloride BP and the pH of the resulting solution is adjusted to pH 3.5 with diluted hydrochloric acid BP After that, the volume was adjusted using injectable sodium chloride BP and mixed thoroughly. The solution was filled in 5 ml type I ampoule made of clear glass, sealed in the upper lattice of the air by dissolving the glass, and sterilized by autoclave at 120 캜 for 15 minutes or longer to prepare an injection solution.

&Lt; Formulation Example 2 > Production of food

Foods containing the topical extract of turmeric or coriander according to the present invention, its nonpolar organic solvent fraction, were prepared as follows.

<2-1> Production of flour food

The topical extract of turmeric or coriander according to the present invention, 0.1 to 10.0 parts by weight of the non-polar organic solvent fraction thereof, is added to wheat flour, and bread, cake, cookies, crackers and noodles are prepared by using the mixture, Food was prepared.

<2-2> Production of soups and gravies

0.1 to 1.0 part by weight of a topical extract of turmeric or coriander according to the present invention and a nonpolar organic solvent fraction thereof were added to soups and juices, and meat processing products for health promotion, soup of noodles and juices were prepared by a conventional method.

<2-3> Preparation of ground beef

10 parts by weight of the topical extract of turmeric or coriander according to the present invention and its nonpolar organic solvent fraction were added to ground beef to prepare ground beef for health promotion by a conventional method.

<2-4> Manufacture of dairy products

0.1 to 1.0 part by weight of a topical extract of turmeric or coriander according to the present invention and its non-polar organic solvent fraction were added to milk, and a variety of dairy products such as butter and ice cream were prepared using the milk in a usual manner.

&Lt; 2-5 >

Brown rice, barley, glutinous rice, and yulmu were dried by a known method and dried, and the mixture was granulated to a powder having a particle size of 60 mesh. Black soybeans, black sesame seeds, and perilla seeds were steamed and dried by a conventional method, and then they were prepared into powder having a particle size of 60 mesh by a pulverizer. The ground-based extract of turmeric or coriander according to the present invention and its nonpolar organic solvent fraction were reduced in pressure in a vacuum concentrator, dried by spraying and dried in a hot-air drier, and pulverized to a size of 60 mesh with a pulverizer to obtain a dried powder.

The above-prepared grains, seeds, and the topical extract of turmeric or coriander according to the present invention, and the dried powders of the non-polar organic solvent fractions thereof were mixed in the following proportions and prepared by a conventional method.

(30 parts by weight of brown rice, 15 parts by weight of yulmu, 20 parts by weight of barley)

Seeds (7 parts by weight of perilla, 8 parts by weight of black beans, 7 parts by weight of black sesame seeds)

Dried powder (1 part by weight) of its non-polar organic solvent fraction,

Ganoderma lucidum (0.5 parts by weight)

(0.5 parts by weight)

&Lt; Formulation Example 3 > Preparation of beverage

A beverage containing a topical extract of turmeric or coriander according to the present invention, its non-polar organic solvent fraction, was prepared as follows.

<3-1> Production of health drinks

(Such as liquid fructose (0.5%), oligosaccharides (2%), sugar (2%), salt (0.5%) and water (75%) and the topical extract of turmeric or coriander according to the present invention, its nonpolar organic solvent fraction Were uniformly blended and instant sterilized, and then packaged in small containers such as glass bottles and plastic bottles to produce health drinks.

<3-2> Preparation of vegetable juice

The vegetable juice for health promotion was prepared by adding 0.5 g of the topical extract of turmeric or coriander according to the present invention to 1,000 ml of vegetable juice such as tomato or carrot.

<3-3> Production of fruit juice

A fruit juice for health promotion was prepared by adding 0.1 g of a topical extract of turmeric or coriander according to the present invention and a non-polar organic solvent fraction thereof to 1,000 ml of fruit juice such as apple or grape.

Claims (9)

Wherein the extract is selected from the group consisting of cancer and inflammatory diseases, wherein the extract is selected from the group consisting of cancerous stem or corn steep liquor, or an ethyl acetate fraction thereof as an active ingredient, and the extract is extracted using any one of C1-C4 lower alcohols A pharmaceutical composition for the prevention or treatment of an IL-6 induced STAT3 mediated disease.
delete The method according to claim 1,
Wherein the extract is extracted with methanol.
delete delete The method according to claim 1,
The cancer is selected from the group consisting of pancreatic cancer, breast cancer, prostate cancer, brain tumor, head and neck carcinoma, melanoma, myeloma, leukemia, lymphoma, liver cancer, gastric cancer, colon cancer, bone cancer, uterine cancer, ovarian cancer, rectal cancer, Wherein the cancer is selected from the group consisting of cancer of the fallopian tube, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, bladder cancer, renal cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma and central nervous system tumor Composition.
The method according to claim 1,
Wherein the inflammatory disease is selected from the group consisting of rheumatoid arthritis, osteoporosis, transglottial hyperplasia, hyperimmunoglobulinemia, anemia, nephritis, cachexia, climacteric disease, angiogenic nephritis, multiple sclerosis, uveitis, chronic thyroiditis, delayed hypersensitivity, contact dermatitis, Pancreatitis, psoriasis, inflammatory idiopathic ataxia, diabetes, and Alzheimer's.
Wherein the extract is selected from the group consisting of cancer and inflammatory diseases selected from the group consisting of extracts of turmeric stalk or corn root, or ethyl acetate fraction thereof, and the extract is extracted using any one of C1-C4 lower alcohols. Functional foods for the prevention or amelioration of induced STAT3 mediated diseases.
delete

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