KR20090109769A - Pharmaceutical composition comprising the extracts, fractions and isolated diterpenoids compounds of Euphorbia kansui as active ingredient - Google Patents

Abstract

PURPOSE: A pharmaceutical composition containing Euphorbia kansui extract, it fraction, and diterpenoid compound is provided to use as an anticancer drug or anti-inflammation agent. CONSTITUTION: A pharmaceutical composition for preventing and treating IL-6 related diseases contains Euphorbia kansui extract, its fraction, and diterpenoid which are isolated from the extract as active ingredient. The Euphorbia kansui extract is isolated using water, low alcohol of C1-C4, or their mixture solvent. The fraction of Euphorbia kansui extract is obtained by concentrating the Euphorbia kansui extract, removing solvent, adding water, alcohol of C1-C4, or their mixture solvent, and fractioning with chloroform, ethylacetate or water in order.

Description

Pharmaceutical composition comprising the extracts, fractions and isolated diterpenoids compounds of Euphorbia kansui as active ingredient}

The present invention is directed to Euphorbia kansui Liou ex Wang) extracts, fractions thereof, or diterpenoids compounds isolated therefrom (diterpenoids) compounds or pharmaceutically acceptable salts thereof as anti-cancer and anti-inflammatory agent containing as an active ingredient.

Signal transducers and activators of transcription (STAT) proteins are DNA-binding proteins that play a role in transcriptional signal transmission and activation. To date, six different factors (STAT1, STAT2, STAT3, STAT4, STAT5 and STAT6) and several isoforms (STAT1-alpha, STAT1-beta, STAT3-alpha and STAT3-beta) have been reported. When the cytokine binds to the receptor, Janus protein tyrosine kinases (JAKs) are activated to phosphorylate STAT, resulting in transition to the nucleus and transcriptional activation of the STAT reactive gene. Events that are mediated by cytokines through STAT activation include cell proliferation and differentiation and prevention of cell death. The specificity of STAT activation is due to specific cytokines, and each STAT responds to a specific cytokine. Growth factors, on the other hand, have also been shown to activate STATs, and binding these factors to cell surface receptors associated with protein tyrosine kinases results in phosphorylation of STATs.

In particular, STAT3 (also Acute Phase Response Factor (APRF), STAT3-alpha) is not only expressed by interleukin-6 (IL-6) but also by epidermal growth factor (EGF). It was found to be activated (Darnell JE et al . , Science 264: 1415-1421, 1994), and has also been reported to play an important role in signal transduction by interferon (Yang CH et. al . , PNAS USA 95: 5568-5572, 1998). In addition, extracellular signal-regulated kinase (ERK2) induces serine phosphorylation and is associated with STAT3 (Jain N et. al . , Oncogene 17: 3157-3167, 1998).

STAT3 is also expressed in most cell types (Zhong Z et al . , PNAS USA 91: 4806-4810, 1994), which induce the expression of genes involved in tissue damage and response to inflammation, and reported to prevent cell death through the expression of Bcl-2 (B-cell leukemia / lymphoma 2) Fukada T et al . , Immunity 5: 449-460, 1996). That is, aberrant or constitutive expression of STAT3 is associated with a number of disease processes. Structural activation and / or overexpression of STAT3 is involved in many forms of cancer, including myeloma, breast carcinoma, prostate cancer, brain tumor, head and neck carcinoma, melanoma, leukemia and lymphoma, especially chronic myeloid leukemia and multiple myeloma (Niu et. al . , Cancer Res 59: 5059-5063, 1999). Specifically, breast cancer cell lines that overexpress EGFR structurally express phosphorylated STAT3 (Sartor CT et al. al . , Cancer Res 57: 978-987, 1997; Garcia R et al . , Cell Growth and Differentiation 8: 1267-1276, 1997). In addition, cells derived from both rat and human prostate cancers have been found to have structurally activated STAT3, and STAT3 activation correlates with malignant potential. Expression of dominant-negative STAT3 has been reported to significantly inhibit the growth of human prostate cells (Ni et al . , Cancer Res 60: 1225-1228, 2000), where STAT3 is associated with some acute leukemias (Gouilleux-Gruart V et al . , Leuk Lymphoma 28: 83-88, 1997) and T cell lymphoma (Yu CL et al . , J Immunol 159: 5206-5210, 1997). Interestingly, STAT3 is structurally phosphorylated on serine residues in chronic lymphocytic leukemia (Frank DA et al. al . , J Clin Invest 100: 3140-3148, 1997), in both myeloid mononuclear cells and cultures from patients with multiple myeloma, STAT-3 has been reported to be structurally active in myeloma tumor cells. These myeloid mononuclear cells are resistant to Fas-mediated apoptosis and express high levels of Bcl-xL. That is, STAT3 signaling has been reported to be essential for the survival of myeloma tumor cells by conferring resistance to apoptosis (Catlett-Falcone R et al . , Immunity 10: 105-115, 1999).

Therefore. The present inventors supervised (Euphorbia kansui Liou ex Wang) extract, fractions thereof and diterpenoid compounds isolated therefrom have completed the present invention by confirming that the inhibitory effect of STAT3 activity can be effectively used for the prevention and treatment of IL-6 related diseases.

An object of the present invention take (Euphorbia kansui Liou ex Wang) extract, fractions thereof, or diterpenoid-based compounds isolated therefrom, or pharmaceutical compositions for the prevention and treatment of IL-6-related diseases containing a pharmaceutically acceptable salt thereof as an active ingredient To provide.

Another object of the present invention is to provide an anti-inflammatory or anticancer agent containing a sensitized extract, a fraction thereof or diterpenoid compounds separated therefrom or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention is to provide a method for preventing and treating IL-6 related diseases using a sensitized extract, a fraction thereof, or a diterpenoid compound isolated therefrom.

Another object of the present invention is to provide a health food for the prevention and improvement of IL-6-related diseases containing water extract, fractions thereof or diterpenoid compounds isolated therefrom.

In order to achieve the above object, the present invention is susceptible to Euphorbia kansui Liou ex Wang) extract, fractions thereof, or diterpenoids compounds isolated therefrom, or IL-6 containing at least one selected from the group consisting of pharmaceutically acceptable salts thereof as an active ingredient It provides a pharmaceutical composition for the prevention and treatment of diseases.

The present invention also provides an anti-inflammatory or anticancer agent containing at least one selected from the group consisting of the sensitized extract, fractions thereof, or diterpenoid compounds separated therefrom or pharmaceutically acceptable salts thereof as an active ingredient. do.

In addition, the present invention provides IL- comprising administering to the subject one or more selected from the group consisting of the sensitized extract, fractions thereof, or diterpenoid compounds isolated therefrom or pharmaceutically acceptable salts thereof. 6 Provide methods for the prevention and treatment of related diseases.

In addition, the present invention provides a method for the prevention of IL-6-related diseases containing at least one selected from the group consisting of the sensitized extract, fractions thereof, or diterpenoid compounds separated therefrom or pharmaceutically acceptable salts thereof. Provide health food for improvement.

Hereinafter, the present invention will be described in detail.

A sensitized extract, a fraction thereof, or a diterpenoid-based compound separated therefrom, which is an active ingredient of the present invention, is prepared by a separation and purification method comprising the following steps:

1) extracting sensitized water with an extraction solvent;

2) filtering and concentrating the water extract of step 1);

3) further fractionating the concentrate of step 2) with an organic solvent;

4) obtaining the active fraction from the fraction of step 3) by column chromatography; And

5) separating the active compound from the active fraction of step 4) by HPLC method.

In the above production method, the supervision of step 1) can be used without limitation, such as cultivated or commercially available, can be used all the above-ground parts, roots, seeds of the tree.

In the above production method, the extraction solvent of step 1) is characterized in that the water, alcohol or a mixed solvent thereof. As the alcohol, C ₁ to C ₄ lower alcohols are preferably used, and as lower alcohols, ethanol or methanol is preferably used. During the extraction, the solvent is preferably extracted by adding 2 to 20 times the amount of water subtraction, and more preferably, 5 times to add the extract. The extraction method uses a method such as hot water extraction, cold needle extraction, reflux cooling extraction or ultrasonic extraction, preferably cold needle extraction is not limited thereto. The temperature of the solvent at the time of extraction is preferably 20 ~ 100 ℃, more preferably room temperature, but is not limited thereto. In addition, the extraction time is preferably 12 hours to 10 days, more preferably 7 days is not limited thereto. In addition, the number of extraction is preferably 1 to 5 times, more preferably three times extraction is not limited thereto. The extract was filtered and concentrated by the above method, and the filtrate was concentrated and lyophilized to obtain an extract.

In the preparation method, the organic solvent of step 3) may be used, such as chloroform, acetate, hexane, dichloromethane, acetone or acetonitrile.

In the above method, the column chromatography of step 4) is separated by performing column chromatography using a filler selected from the group consisting of silica gel, Sephadex, RP-18, polyamide, Toyopearl and XAD resin. It can be purified. Column chromatography can be carried out several times by selecting the appropriate filler as required. In the embodiment of the present invention, the silica gel was used as the filler in the column chromatography, and the active fraction was first separated using a step gradient gradient system in which the concentration of methanol to chloroform was increased at a ratio of 100: 0 to 1: 1. The fractions with the highest IL-6-induced luciferase inhibitory activity were collected and the active fractions were collected using a step gradient gradient system in which the concentration of methanol in water was increased by 10% from 50% to 100%. 6 induced luciferase inhibitory activity was able to obtain an active fraction (see Table 1 and Table 2).

In the above method, the active compound was separated through the material separated in 15 minutes and 30 minutes by using 70% methanol as an elution solvent in HPLC (Phenomenex Luna 10 μm C ₁₈ column) of step 5). Could be obtained. Molecular weight measurement, molecular formula estimation, and nuclear magnetic resonance (NMR) confirmed the structure of the material separated into the 15 and 30 components, and the material separated into the 15 components was a diterpenoid compound. Kansuinine A (Kansuinine A) represented by Chemical Formula 1, and the substance separated in 30 minutes was found to be Kansuinine B (Kansuinine B) represented by Chemical Formula 2, which is a diterpenoid compound.

The present invention provides a method for preventing IL-6-related diseases containing at least one selected from the group consisting of sensitized extracts, fractions thereof or diterpenoid compounds isolated therefrom or pharmaceutically acceptable salts thereof as an active ingredient, and Provided is a therapeutic pharmaceutical composition.

In a specific embodiment of the present invention, it was confirmed that the ethanol extract of the present invention, the chloroform fraction thereof, or the diterpenoid compound isolated therefrom inhibited IL-6 induced luciferase activity (see FIGS. 1 to 3). In particular, the sensitized ethanol extract and the chloroform fraction inhibited at least 50.4% and at least about 75% IL-6 induced luciferase at concentrations of 50 μg / ml, respectively (see FIGS. 1 and 2). In addition, the diterpenoid compounds of Formulas 1 and 2 derived from the chloroform fractions had IC ₅₀ values of 2.35 and 5.91 μg / ml, respectively (see FIG. 3). In addition, the compounds represented by Formulas 1 and 2 of the present invention also showed IL-6 induced STAT3 phosphorylation inhibitory activity (see FIG. 4). According to the above results, the sensitized extract of the present invention, fractions thereof, or diterpenoid compounds isolated therefrom exhibit STAT3 inhibitory activity, and thus may be usefully used in pharmaceutical compositions for preventing and treating IL-6 related diseases.

The water extract is prepared by extracting with water, alcohol or a mixed solvent thereof, the alcohol is characterized in that the C ₁ to C ₄ lower alcohol, such as methanol, ethanol or butanol. The water extract may be prepared using a method such as hot water extraction, cold needle extraction, reflux cooling extraction or ultrasonic extraction, preferably by cold needle extraction.

The fractions are concentrated in the sensitized extract to completely remove the solvent, suspended in water, alcohol of C ₁ ~ C ₄ , or a mixed solvent thereof, chloroform fraction or ethyl acetate obtained by sequentially fractionating with chloroform, ethyl acetate and water Fraction. Of the above fractions, the chloroform fraction is preferred. This process may use a conventional fractional extraction method, preferably a fractional funnel.

The diterpenoid-based compound may be Kansuinine A represented by Formula 1 or Kansuinine B represented by Formula 2. The diterpenoid compounds may be used in the form of a pharmaceutically acceptable salt, and include all salts, hydrates, and solvates prepared by conventional methods. The diterpenoid compounds of the present invention can be extracted and separated from water, synthetically prepared in a conventional organic synthesis method, and commercially available reagents can be used. Specifically, the diterpenoid compound is a mobile phase with a chloroform / methanol (100: 0 to 1: 1) mixed solvent with respect to the fraction, and subjected to silica gel column chromatography to obtain an active fraction primarily. A water-methanol (50: 50-0: 100) mixed solvent was used as the mobile phase for the first active fraction, and reverse phase column chromatography was performed to obtain the second active fraction, followed by 70% of the second active fraction. It can be obtained by pure separation of the compound eluting at 15 and 30 minutes by using methanol as the mobile phase and performing high performance liquid chromatography.

The IL-6-related disease is characterized in that any one selected from the group consisting of inflammatory disease, leukemia and cancer. Specifically, the disease may be selected from the group consisting of inflammatory diseases, particularly rheumatoid arthritis, and cancers including myeloma, uterus, prostate, head and neck, and brain cancers, myeloma, melanoma, leukemia, lymphoma, etc. It is not limited.

 In the present invention, the pharmaceutically acceptable salt is an addition salt formed by free acid. Suitable free acids may be organic and inorganic acids, inorganic acids may be hydrochloric acid, bromic acid, sulfuric acid and phosphoric acid, and organic acids may be citric acid, acetic acid, lactic acid, tartariac acid, maleic acid, Fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, galluxuronic acid, embonic acid, glutamic acid or aspartic acid Etc. can be used. Furthermore, the sensitized extract, fractions thereof or diterpenoid compounds isolated therefrom according to the present invention are not only pharmaceutically acceptable salts, but also all salts, hydrates and solvates that can be prepared by conventional methods. It may include.

The pharmaceutical composition of the present invention may optionally contain one or more from sensitized extract, fractions thereof, or diterpenoid-based compounds isolated therefrom or pharmaceutically acceptable salts thereof, and may be the same as or in addition to the above components. It may contain one or more active ingredients exhibiting similar functions.

The pharmaceutical composition of the present invention comprises 0.0001 to 10% by weight, preferably 0.001 to 1% by weight of the sensitized extract, fractions thereof, or diterpenoid compounds separated therefrom based on the total weight of the composition.

The pharmaceutical composition according to the present invention may further include conventionally used carriers, excipients, disintegrants, sweeteners, lubricants, flavoring agents and diluents. The carrier, excipient and diluent may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The disintegrants include sodium starch glycolate, crospovidone, croscarmellose sodium, alginic acid, calcium carboxymethyl cellulose, sodium carboxymethyl cellulose, chitosan, guar gum, low-substituted hydroxypropyl cellulose, magnesium aluminum silicate, and polyacryline Potassium and the like. In addition, the pharmaceutical composition according to the present invention may further include a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, Calcium hydrogen phosphate, lactose, mannitol, malt, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, opadry, sodium starch glycolate, carnauba lead, synthetic aluminum silicate, stearic acid, magnesium stearate, Aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol, talc and the like can be used. The pharmaceutically acceptable additive according to the present invention is preferably included 0.1 to 90 parts by weight based on the pharmaceutical composition.

In addition, the subject to which the pharmaceutical composition of the present invention can be applied is a vertebrate and preferably a mammal, more preferably an experimental animal such as a rat, rabbit, guinea pig, hamster, dog, cat, and most preferably It is ape-like, such as chimpanzees and gorillas. In addition, the pharmaceutical composition of the present invention can be administered orally or parenterally, and intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine epidural injection, cerebrovascular (intracerebroventricular) ) Or by intrathoracic injection. In addition, the pharmaceutical composition may be used alone or in combination with methods of using surgery, hormonal therapy, drug treatment, and biological response modifiers for the prophylaxis and treatment of IL-6 related diseases.

The preferred dosage of the pharmaceutical composition of the present invention depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may 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 0.0001 to 100 mg / kg, preferably at 0.001 to 100 mg / kg. Administration may be administered once a day or may be divided several times.

The present invention also provides an anti-inflammatory or anticancer agent containing at least one selected from the group consisting of the sensitized extract, fractions thereof, or diterpenoid compounds separated therefrom or pharmaceutically acceptable salts thereof as an active ingredient. do.

In a specific embodiment of the present invention, it was confirmed that the ethanol extract of the present invention, the chloroform fraction thereof, or the diterpenoid compound isolated therefrom inhibited IL-6 induced luciferase activity (see FIGS. 1 to 3). In addition, compounds represented by Formulas 1 and 2 of the present invention also showed IL-6 induced STAT3 phosphorylation inhibitory activity (see FIG. 4). According to the above results, the sensitized extract of the present invention, fractions thereof, or diterpenoid compounds separated therefrom exhibit STAT3 inhibitory activity, and thus can be usefully used for anti-inflammatory or anticancer agents.

The sensitized extract of the present invention, fractions thereof, or diterpenoid compounds separated therefrom or pharmaceutically acceptable salts thereof are powders, granules, tablets, capsules, suspensions, emulsions, syrups, respectively, according to conventional methods. Oral dosage forms, external preparations, suppositories, and sterile injectable solutions.

Solid preparations for oral administration include powders, granules, tablets, capsules, soft capsules, pills and the like. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have. Formulations for parenteral administration include powders, granules, tablets, capsules, sterile aqueous solutions, solutions, non-aqueous solutions, suspensions, emulsions, syrups, suppositories, aerosols, etc. It may be used in the form of a formulation, and preferably, an external skin pharmaceutical composition of cream, gel, patch, spray, ointment, warning agent, lotion agent, linen agent, pasta agent or cataplasma agent may be prepared and used. It is not limited to this. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

In addition, the present invention is IL- comprising the step of administering to the subject one or more selected from the group consisting of the sensitized extract, fractions thereof, or diterpenoid compounds separated therefrom or pharmaceutically acceptable salts thereof. 6 Provide methods for the prevention and treatment of related diseases.

The subject is a vertebrate and preferably a mammal, more preferably an experimental animal such as a rat, rabbit, guinea pig, hamster, dog, cat, more preferably a mammal other than a human, most preferably It is ape-like, such as chimpanzees and gorillas.

In addition, the IL-6-related disease is characterized in that any one selected from the group consisting of inflammatory disease, leukemia and cancer. Specifically, the IL-6-related disease may be selected from the group consisting of inflammatory diseases, particularly rheumatoid arthritis, and cancer, myeloma, melanoma, leukemia and lymphoma, including breast, uterus, prostate, head and neck, and brain cancers. May be, but is not limited thereto.

In addition, the sensitized extract of the present invention, fractions thereof, or diterpenoid-based compounds isolated therefrom or pharmaceutically acceptable salts thereof may be orally or parenterally administered, and may be intraperitoneally injected or rectally administered parenterally. It can be administered by internal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrauterine dural injection, intracerebroventricular injection or intrathoracic injection. In addition, the sensitized extract of the present invention, fractions thereof, or diterpenoid-based compounds isolated therefrom or pharmaceutically acceptable salts thereof may be used alone or in the subject for the prevention and treatment of IL-6 related diseases. It can be used in combination with methods using surgery, hormonal therapy, drug therapy and biological response modifiers.

Preferred dosages of the sensitized extract of the present invention, fractions thereof, or diterpenoid-based compounds isolated therefrom or pharmaceutically acceptable salts thereof include the condition and weight of the patient, the extent of the disease, the form of the drug, the route and duration of the patient. Depending on, it can be appropriately selected by those skilled in the art. However, for the desired effect, the above-mentioned persimmon extract, fractions thereof or diterpenoid compounds or pharmaceutically acceptable salts thereof of the present invention are 0.0001 to 100 mg / kg per day, preferably Administration at 0.001 to 100 mg / kg is preferred. Administration may be administered once a day or may be divided several times.

In addition, the present invention provides a method for the prevention of IL-6-related diseases containing at least one selected from the group consisting of the sensitized extract, fractions thereof, or diterpenoid compounds separated therefrom or pharmaceutically acceptable salts thereof. Provide health food for improvement.

The sensitized extract of the present invention, fractions thereof, or diterpenoid-based compounds separated therefrom or pharmaceutically acceptable salts thereof may be added to foods or used together with other foods or food ingredients, and used in conventional methods. Can be used accordingly. The mixing amount of the active ingredient can be suitably determined according to the purpose of use (prevention or improvement). Generally, in the preparation of food or beverages, the composition of the present invention is added at 0.2 to 20% by weight, preferably 0.24 to 10% by weight relative to the raw materials. However, in the case of long-term intake for health and hygiene or health control purposes, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.

The health food of the present invention may contain various flavors or natural carbohydrates as additional ingredients. The above-mentioned natural carbohydrates are glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, synthetic sweetening agents such as saccharin, aspartame, and the like can be used. The ratio of the natural carbohydrate is preferably selected in the range of 0.01 to 0.04 parts by weight, preferably about 0.02 to 0.03 parts by weight, per 100 parts by weight of the health food of the present invention.

There is no particular limitation on the kind of food. Examples of foods to which the above-mentioned substances may be added include dairy products including drinks, meat, sausages, breads, biscuits, rice cakes, chocolates, candy, snacks, confectionery, pizza, ramen, other noodles, gums and ice cream, various soups, Beverages, alcoholic beverages, vitamin complexes, and the like, and include all healthy foods in the conventional sense.

In addition to the above, the health food of the present invention includes various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols. And carbonation agents used in carbonated beverages. In addition, the health food of the present invention may contain a flesh for producing natural fruit juice, fruit juice beverage and vegetable beverage. These components can be used independently or in combination. Although the ratio of such an additive is not critical, it is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health food of the present invention.

Reduction of the Invention ( Euphorbia) kansui Liou ex Wang) extract, fractions and diterpenoid compounds isolated therefrom can be usefully used as anticancer and anti-inflammatory agents by inhibiting STAT3 activation induced by IL-6.

The present invention is described in more detail based on the following examples, experimental examples and preparation examples, but the present invention is not limited thereto.

Example 1 Preparation of Water-Resistant Ethanol Extracts

Reduction ( Euphorbia kansui Liou ex Wang; Moorim dry materials wholesale, Daejeon) was washed with water, dried in the shade and then used by grinding. It was immersed in 100% ethanol three times the weight of the sensitized weight dried on 1.2 kg of sensitized powder, extracted at room temperature for 7 days and then filtered through a filter paper. The extract was concentrated using a concentrator under reduced pressure until the solution completely evaporated to give a dried crude extract. As a result of using the above method, 105 g of extract could be prepared from sensitized powder (1.2 kg).

<Example 2> Preparation accept fraction

The ethanol extract obtained by the method of Example 1-1 was suspended in distilled water, and then systematically fractionated in the order of chloroform, ethyl acetate and water. After repeated three times, each solvent fraction was concentrated under reduced pressure.

As a result, solvent fractions of chloroform (18 g), ethyl acetate (7 g) and water (80 g) were obtained, respectively.

< Example  3> active Fraction  Produce

An active fraction was obtained using the IL-6 induced luciferase inhibitory activity as an index among the fractions obtained from the sensitized extract.

<3-1> Chlorform Methanol Activity Fraction

The chloroform fraction (18 g) obtained by the method of Example 2 having the highest IL-6 induced luciferase inhibitory activity among the sensitized fractions was composed of chloroform: methanol = (100/0 to 1/1). Each of the 21 small fractions was eluted sequentially using column chromatography (70-230 mesh, Merck) of silica gel using a gradient solvent system.

IL-6-induced luciferase inhibitory activity was measured according to the method of Experimental Example 1 among the active fractions, and an active fraction having the highest activity (2 g, chloroform: concentration of methanol = 20: 1 fraction) was obtained (Table 1). One).

Column prototyping conditions (sample concentration 50 μg / ml) Fraction Mobile phase Chloroform: Methanol Inhibition% 1-3 100: 0 12.9 4-6 50: 1 35.3 7-9 20: 1 81.7 10 -12 10: 1 42.9 13-15 5: 1 1.3 16-18 2: 1 3.6 19-21 1: 1 2.8

<3-2> methanol activity Fraction

Chloroform obtained by the above method: Among the methanol fractions, the fractions with the highest inhibitory activity were collected and reversed phase column chromatography using 50%, 60%, 70%, 80%, 90%, 100% methanol as the elution solvent. Phase column chromatography, ODS gel; YMC-Gel RP-18, 70-230 mesh) were used to elute each of the 30 small fractions sequentially.

IL-6-induced luciferase inhibitory activity was measured according to the method of Experimental Example 1 among the active fractions, and an active fraction having the highest activity (800 mg, water: methanol concentration = 30:70 fraction) was obtained (Table 2).

Column prototyping conditions (sample concentration 50 μg / ml) Fraction Mobile phase water Methanol Inhibition% 1-5 50% 50% 20.3 6-10 40% 60% 35.2 11-15 30% 70% 87.9 16-20 20% 80% 47.3 21-25 10% 90% 12.3 26-30 0% 100% 3.0

< Example  4> Isolation of the Compound

Among the active fractions obtained by the method of Example 3, the fractions having the highest inhibitory activity were collected, and 70% methanol was flowed at 7 ml / min as an eluting solvent, followed by high performance liquid chromatography; HPLC, YMC Jsphere ODS H-80 (250 × 20 mm)] gave pure compounds. Detection of the compounds was carried out at UV 210 nm, the IL-6 induced luciferase inhibitory activity of the pure compounds was measured according to the method of Experimental Example 1, the IL-6 induced luciferase inhibitory active compound was 15 minutes and 30 Eluted in minutes.

< Example  5> IL -6 induction Luciferase  Structural Analysis of Inhibitory Active Compounds

<5-1> Structural analysis of the compound eluted at 15 minutes

The compound eluted at 15 minutes separated by the method of Example 4 was colorless crystals, and the molecular weight was measured by high resolution ESI-MS (Navigator). As a result, [M + Na] + was m / z 755.7 and HRFAB-MS ( HX 110A / HX 100A spectrometer, JEOL, Japan) estimated the molecular formula C ₃₇ H ₄₆ O ₁₅ . Maximum absorption was found at 230 nm as measured by a spectrophotometer (model 8453 spectrophotometer, Packard, USA). During the Varian Unity 300 spectrometer, USA (NMR) test to determine the structure of the compound, 37 carbons estimated in the mass spectrometry on ¹³ C-NMR were observed. ^One terminal olefin proton (δ 5.17) was observed in the ¹ H-NMR spectrum, and two methylene protons (δ 2.20, 2.23) were observed. In addition, methyl protons (δ 1.91, 1.98, 2.07, 2.09, 2.18) observed in five acetyl groups and protons (δ 7.42, 7.53, 8.03) observed in one benzene group were found, and four terminal methyl protons (δ 0.92). , 1.14, 1.29, 1.30).

The compound is published in Li-Yan Wang et. al . , J Nat Prod 65: 1246-1251, 2002) After a comparison with the data of, it turned out to be kansu compounds of the formula A coix seed (Kansuinine A).

<Formula 1>

.

.

<5-2> Structural analysis of the compound eluted at 30 minutes

Example how the compounds eluted in the 30 minutes separated by 4 is a result of measuring the molecular weight as colorless crystals, [M + Na] + a m / was z 746.7, is in mounds by ESI-MS molecular formula C ₃₈ H ₄₂ O ₁₄ was estimated. Ultraviolet absorbance of the compound was measured and the maximum absorption was found at 232 nm. During the NMR test to determine the structure of the compound, 38 carbons were observed in ¹³ C-NMR. ^One terminal olefinic proton (δ 5.14) was observed in the ¹ H-NMR spectrum, and protons (δ 1.89, 2.31) and two benzene group protons (δ 6.92, 7.02, 7.11, 7.22, 7.50, 7.55). In addition, methine protone (δ 2.52, 3.54, 3.92) bonded to one methylene proton (δ 2.21) and three hydroxyl groups was observed.

Such compounds are described in Li-Yan Wang et. al . , J Nat Prod 65: 1246-1251, In comparison with the data of 2002), the compound was identified as Kansuinine B.

<Formula 2>

.

.

Experimental Example 1 IL- 6 Induced Luciferase Inhibitory Activity Assay

<1-1> Transformant Preparation

Dispense HepG2 cells (ATCC HB-8065) at 5 × 10 ⁴ cells / well in 96 well plates, then 10% FBS (v / v), 60.0 mg / l kanamycin sulfate (Kamyamycin sulfate; Gibco., USA) and 2.0 DMEM culture medium containing g / l sodium hydrogen carbonate (NaHCO ₃ ; Sigma, USA) was used to incubate at 37 ° C. with 5% CO ₂ until 80% confluent in the culture dish. Subsequently, 50 μl of serum-free medium was exchanged, and a mixture of 0.1 μg pSTAT3-TA-Luc (Clontech, Calif.) And 0.3 μl lipofectamin reagent (Invitrogen, USA) was added to each well and reacted for 3 hours. -TA-Luc was transfected and replaced with freshly prepared 200 μl DMEM culture medium and incubated for an additional 24 hours.

<1-2> IL -6 reactivity STAT3  Reporter Genetic Test

The transfected cells were cultured serum-free (serum starvation) with 1% BSA / DMEM, and the samples were treated for 1 hour as follows, followed by incubation for 3 hours with the addition of 10 ng / ml IL-6 (R & D system, USA). .

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

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

3: ethanol extract (50 μg / ml);

4: water-sensitive chloroform fraction (50 μg / ml);

5: compound 1 (3.12, 6.25, 12.5, 25, 50 μg / ml); And,

6: compound 2 (3.12, 6.25, 12.5, 25, 50 μg / ml).

The reaction cells were washed with PBS, 50 μl lysis buffer (luciferase assay system, promega, USA) was added thereto, stirred for 1 minute, and then 30-100 μl luciferase substrate (luciferase assay system, promega, USA) was added thereto. The color development was measured within 5 minutes with a luminometer (EG & G BERTHOLD, USA).

As a result, the supernatant ethanol extract inhibited 50.4% of IL-6 induced luciferase at a concentration of 50 μg / ml (FIG. 1). In addition, the chloroform fraction fractionated therefrom inhibited at least about 75% of IL-6 induced luciferase at a concentration of 50 μg / ml (FIG. 2). In addition, the diterpenoids compounds of Formulas 1 and 2 derived from the chloroform fractions had IC ₅₀ values of 2.35 and 5.91 μg / ml, respectively (FIG. 3).

Experimental Example 2 STAT3 Phosphorylation Inhibitory Activity Induced by IL- 6

HepG2 cells were dispensed at 5 × 10 ⁴ cells / well in 6-well plates and cultured 80% full in a culture dish, then exchanged with serum-free medium for an additional 6 hours, and the samples were treated for 30 minutes as follows.

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

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

3: IL-6 treated group (20 ng / ml);

4: compound 1 treatment group (0.02, 0.09, 0.39, 1.56 and 6.25 μg / ml); And,

5: compound 2 treated group (0.09, 0.39, 1.56, 6.25 and 25.00 μg / ml).

After reacting for 10 minutes with 20 ng / ml IL-6, 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, 20 mg / ml aprotonin; Sigma, USA] and the cells were lysed and centrifuged (13000 g, 15 minutes) to obtain a supernatant with dissolved protein. At this time, HepG2 cells not treated with the sample and IL-6 were used as a control. Protein concentration was quantified using a DC protein test kit (Bio-Rad, USA), and electrophoresed at 30 mA for 2 hours by loading the protein on a 10% SDS polyacrylamide gel (SDS-PAGE). After electrophoresis, the protein of the gel was transferred to a PVDF membrane (Westran® S, pore size 0.2 mm; Whatman, USA) at 90 V for 90 minutes. The transcribed 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) for 12 hours at 4 ° C and T Washed 5 times with TBS. The membrane was treated with polyclonal antibody of phospho-STAT3 (1: 1000 dilution) as a primary antibody for 2 hours. After washing 5 times with T-TBS, HRP-binding anti-rabbit 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 the dark.

As a result, as shown in Figure 4 compounds represented by Formulas 1 and 2 of the present invention showed IL-6 induced STAT3 phosphorylation inhibitory activity.

< Production Example  1> Preparation of Pharmaceutical Formulations

1. Preparation of powder

Persimmon extract of the present invention, fractions thereof or diterpenoid compounds isolated therefrom or pharmaceutically acceptable salts thereof 2 g

1 g lactose

The above ingredients were mixed and filled in airtight cloth to prepare a powder.

2. Preparation of Tablets

100 mg of sensitized extract of the present invention, fractions thereof or diterpenoid compounds isolated therefrom or pharmaceutically acceptable salts thereof

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

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

3. Preparation of Capsule

100 mg of sensitized extract of the present invention, fractions thereof or diterpenoid compounds isolated therefrom or pharmaceutically acceptable salts thereof

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

4. Manufacture of rings

Persimmon extract of the present invention, fractions thereof or diterpenoid compounds isolated therefrom or pharmaceutically acceptable salts thereof 1 g

Lactose 1.5 g

1 g of glycerin

Xylitol 0.5 g

After mixing the above components, it was prepared to be 4 g per ring in a conventional manner.

5. Preparation of Granules

150 mg of sensitized extract of the present invention, fractions thereof or diterpenoid compounds isolated therefrom or pharmaceutically acceptable salts thereof

Soybean Extract 50mg

Glucose 200 mg

Starch 600 mg

After mixing the above components, 100 mg of 30% ethanol was added and dried at 60 ° C. to form granules, and then filled in fabric.

Preparation Example 2 Preparation of Food

Food products containing the sensitized extract of the present invention, fractions thereof, or diterpenoid compounds separated therefrom or pharmaceutically acceptable salts thereof were prepared as follows.

1. Preparation of Cooking Seasonings

20-95 parts by weight of the sensitized extract of the present invention, fractions thereof, or diterpenoid compounds separated therefrom or pharmaceutically acceptable salts thereof were prepared for cooking for health promotion.

2. Manufacturing of Flour Foods

Persimmon extract of the present invention, fractions thereof, or diterpenoid compounds separated therefrom, or 0.5 to 5.0 parts by weight of a pharmaceutically acceptable salt thereof are added to flour, and the mixture is used to make bread, cake, cookies, crackers. And noodles were prepared to prepare foods for health promotion.

3. Manufacture of dairy products

5 to 10 parts by weight of the persimmon extract of the present invention, fractions thereof, or diterpenoid compounds separated therefrom or pharmaceutically acceptable salts thereof are added to milk, and various dairy products such as butter and ice cream using the milk Was prepared.

4. Manufacture of wire

Brown rice, barley, glutinous rice, and yulmu were alphanated by a known method to distribute the dried ones, and then prepared into a powder having a particle size of 60 mesh.

Black beans, black sesame seeds, and perilla were also steamed and dried by a known method, and then ground to a powder having a particle size of 60 mesh.

The grains, seeds and the dry extract of the present invention and the polysaccharide fraction were prepared by blending the powders in the following ratio.

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

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

Persimmon extract of the present invention, fractions thereof or diterpenoid compounds isolated therefrom or pharmaceutically acceptable salts thereof (3 parts by weight),

Ganoderma lucidum (0.5 parts by weight),

Foxglove (0.5 part by weight)

< Production Example  3> Manufacture of beverage

1. Manufacture of health drinks

       Persimmon extract of the present invention, fractions thereof or diterpenoid compounds isolated therefrom or pharmaceutically acceptable salts thereof 1000 mg

       Citric acid 1000 mg

       100 g oligosaccharides

       Plum concentrate 2 g

       1 g of taurine

       Add 900 ml of purified water

After mixing the above components according to the conventional healthy beverage manufacturing method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and then refrigerated and stored Used to prepare the healthy beverage composition of the invention.

Although the composition ratio is a composition suitable for a preferred beverage in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and intended use.

1 is a diagram showing the expression inhibitory activity of IL-6 induced luciferase of water ethanol extract.

Figure 2 is a diagram showing the expression inhibitory activity of IL-6 induced luciferase of water-sensitive chloroform fraction.

3 is a diagram showing the expression inhibitory activity of IL-6 induced luciferase of the diterpenoid compounds of Formulas 1 and 2 according to the present invention.

Figure 4 is a diagram showing the IL-6 induced STAT3 phosphorylation inhibitory activity of the diterpenoid compounds represented by formulas (1) and (2).

Claims (18)

Reduction ( Euphorbia kansui IL-6-related diseases containing at least one selected from the group consisting of Liou ex Wang extract, fractions thereof, or diterpenoids compounds isolated therefrom or pharmaceutically acceptable salts thereof Pharmaceutical composition for the prevention and treatment of. The pharmaceutical composition according to claim 1, wherein the water extract is prepared by extracting with water, C ₁ to C ₄ lower alcohols, or a mixed solvent thereof. The pharmaceutical composition according to claim 1, wherein the water extract is prepared by cold extraction. The method of claim 1, wherein the fractions are concentrated by removing the solvent to completely remove the solvent, and then suspended in water, alcohol of C ₁ ~ C ₄ , or a mixed solvent thereof, and sequentially partitioned with chloroform, ethyl acetate and water Pharmaceutical composition, characterized in that the obtained chloroform fraction. The diterpenoid compound of claim 1, wherein the fraction is a chloroform / methanol (100: 0 to 1: 1) mixed solvent with respect to the mobile phase and subjected to silica gel column chromatography to obtain an active fraction primarily. For the obtained primary active fraction, a water: methanol (50: 50 ~ 0: 100) mixed solvent was used as a mobile phase, and reverse phase column chromatography was performed to obtain an active fraction secondarily. Pharmaceutical composition, characterized in that the compound eluted at 15% by performing high-performance liquid chromatography with 70% methanol as a mobile phase. The diterpenoid compound of claim 1, wherein the fraction is a chloroform / methanol (100: 0 to 1: 1) mixed solvent with respect to the mobile phase and subjected to silica gel column chromatography to obtain an active fraction primarily. For the obtained primary active fraction, a water: methanol (50: 50 ~ 0: 100) mixed solvent was used as a mobile phase, and reverse phase column chromatography was performed to obtain an active fraction secondarily. Pharmaceutical composition, characterized in that the compound is eluted in 30 minutes by high-performance liquid chromatography with 70% methanol as a mobile phase. The pharmaceutical composition according to claim 5, wherein the diterpenoid compound is Kansuinine A represented by Chemical Formula 1. <Formula 1>

. The pharmaceutical composition according to claim 6, wherein the diterpenoid compound is Kansuinine B represented by Formula 2: <Formula 2>

. The method of claim 1, wherein the IL-6-related disease consists of inflammatory diseases, particularly rheumatoid arthritis, and cancers including myeloma, uterus, prostate, head and neck, and brain cancers, myeloma, melanoma, leukemia, lymphoma, and the like. Pharmaceutical composition, characterized in that any one selected from the group. The method of claim 1, wherein the sensitizing extract, fractions thereof, or diterpenoid compounds isolated therefrom or pharmaceutically acceptable salts thereof have STAT3 (signal transducers and activators of transcription 3) inhibitory activity Pharmaceutical composition. An anti-inflammatory agent containing at least one selected from the group consisting of sensitized extracts, fractions thereof or diterpenoid compounds separated therefrom or pharmaceutically acceptable salts thereof as an active ingredient. The anti-inflammatory agent according to claim 11, wherein the diterpenoid-based compound is Kansuinine A represented by Formula 1: <Formula 1>

. 12. The anti-inflammatory agent according to claim 11, wherein the diterpenoid compound is Kansuinine B represented by Formula 2: <Formula 2>

. An anticancer agent containing at least one selected from the group consisting of sensitized extracts, fractions thereof or diterpenoid compounds isolated therefrom or pharmaceutically acceptable salts thereof as an active ingredient. 15. The anticancer agent according to claim 14, wherein the diterpenoid compound is Kansuinine A represented by Formula 1: <Formula 1>

. The anticancer agent according to claim 14, wherein the diterpenoid-based compound is Kansuinine B represented by Chemical Formula 2. <Formula 2>

. Prevention of IL-6 related diseases comprising administering to a subject one or more selected from the group consisting of sensitized extracts, fractions thereof or diterpenoid compounds isolated therefrom or pharmaceutically acceptable salts thereof Treatment method. Health food for the prevention and improvement of IL-6-related diseases containing at least one selected from the group consisting of sensitized extract, fractions thereof or diterpenoid compounds isolated therefrom or pharmaceutically acceptable salts thereof.

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