KR101150643B1 - A composition comprising the compounds isolated from the Inulae Flos extract of Inula japonica Thunberg having anti-inflammatory or anti-allergic activity - Google Patents

Abstract

 The present invention relates to a composition for the prophylaxis or treatment of allergic diseases such as inflammation and asthma, which contains a compound isolated from the ectopic extract as an active ingredient, wherein the compound is a cyclooctane from mast cells that cause various inflammation and allergic diseases. It inhibits the production of sigenase-2 (COX-2) dependent prostaglandin and the production of leukotriene and at the same time inhibits the release of histamine from mast cells, thereby preventing and treating various inflammatory or allergic diseases. It can be usefully used as a medicine or health functional food.

Description

 A composition comprising the compounds isolated from the Inulae Flos extract of Inula japonica Thunberg having anti-inflammatory or anti-allergic activity}

 The present invention relates to a composition for preventing or treating allergic diseases such as inflammation and asthma.

Document 1 Murakami M et al, Phospholipase A ₂ enzymes. Prostaglandins Other Lipid Mediat 68-69 , pp.3-58, 2002

Reid RC, Inhibitors of secretory phospholipase A ₂ group IIA. Curr Med Chem 12 , pp. 3011-3026, 2005

Loewen PS, Review of the selective COX-2 inhibitors celecoxib and rofecoxib: focus on clinical aspects. CJEM 4 , pp.268-75, 2002

Ueno A, Inflammation-allergy and prostanoids. (One). Prostanoids in experimental inflammatory reaction. Nippon Yakurigaku Zasshi 117 , pp.255-61, 2001

Lotzer K et al, The 5-lipoxygenase pathway in arterial wall biology and atherosclerosis. Biochim Biophys Acta 1736 (1) , pp.30-7, 2005

Back M, Leukotrienes: potential therapeutic targets in cardiovascular diseases. Bull Acad Natl Med 190 (7) , pp. 1511-1518; discussion pp.1518-1521, 2006

[Ref. 7] Jung-Seop Shin and Min-Kyo Shin, Do Hae Hyang Drug (Medicinal Medicine) 1054-1056, 1998

[Reference 8] Jiangsu New Medical College. A Dictionary of Tranditional Chinese Drug. Sanghai People's Press: Shanghai, pp.324, 1997

Reference 9 Chinese Academy of Medicine. Institute of Pharmacy. Traditional Chinese Medicine, pp. 476, 1984

10. Qin JJ et al, Japonicaones AD, bioactive dimeric sesqiterpenes from Inula japonica Thunb. Bioorg & Med Chem Lett 19 , pp.710-713, 2009

[11] Shan JJ et al, Anti-diabetic and hypolipidemic effects of aqueous-extract from the flower of Inula japonica in alloxan-induced diabetic mice. Biol Pharm Bull 29 , pp.455-459, 2006

12. Jin M et al, The naturally occurring flavolignan, deoxypodophyllotoxin, inhibits lipopolysaccharide-induced iNOS expression through the NF-kappaB activation in RAW264.7 macrophage cells. Biol Pharm Bull 31 (7) , pp. 1312-15, 2008

Son JK et al, Ginkgetin, a Biflavone from Ginko biloba leaves, inhibits cyclooxygenases-2 and 5-lipoxygenase in mouse bone marrow-derived mast cells. Biol Pharm Bull 28 (12) , pp.2181-84, 2005

[14] Yang JH et al, Anti-allergic activity of an ethanol extract from Salviae miltiorrhiza . Arch Pharm Res 31 (12) , pp. 1597-603, 2008

Inflammation is the defense of biological tissue against local injuries. In other words, in response to various harmful stressors, the inflammatory response is a bioprotective response that attempts to remove the injury caused by the stimulus and restore the original state. Inflammatory stimuli include infection or chemical or physical stimuli. Bioconstituents involved in the inflammatory response include low-molecular or high-molecular chemicals such as free radicals, proteins, sugars, and lipids, and plasma, blood cells, blood vessels, and connective tissue. The process of inflammation is usually divided into two, and can be divided into acute and chronic inflammation. Acute inflammation is a short-term reaction within days, and plasma components or blood cells are involved in the removal of foreign bodies through the microcirculation system. Chronic inflammation lasts a long time and shows tissue growth.

Allergic diseases are mainly mediated by mast cells in tissues activated by antigen-antibody reactions and basophils and eosinophils in blood (mainly histamine, leukotrienes, tumor necrosis factor α (TNFα)). , Cytokines, etc.). Since the use of allergy therapeutic drugs currently used remains symptomatic, there is an urgent need for the development of more fundamental therapeutic drugs.

Key mediators that drive inflammatory and allergic diseases prostaglandin acids (prostaglandindes), leukotriene acids (leukotriens), platelet activating factor (PAF), etc. is phospholipase _{A 2 (phospholipase A 2, PLA} 2) and cyclooxygenase Produced from precursor arachidonic acid by cyclooxygenase and lipoxygenase. The arachidonic acid metabolic rate determining enzyme PLA ₂ divides the cell resistance (cPLA ₂₎ and extrinsic cell (secretory PLA _2, sPLA ₂₎ in accordance with the intracellular distribution position, the sPLA ₂ Isoenzyme (isozyme) is 10 kinds, in particular the enzyme sPLA ₂ -IIA, sPLA ₂ -V is known is also involved directly and indirectly to various inflammatory diseases, allergic reactions (Murakami M et al, Phospholipase A ₂ Prostaglandins Other Lipid Mediat 68-69 , pp.3-58, 2002; Reid RC, Inhibitors of secretory phospholipase A ₂ group IIA.Curr Med Chem 12 , pp. 3011-3026, 2005).

Prostaglandins bind to specific cell surface receptors and act to increase the intracellular concentrations of cyclic Adenosine MonoPhosphate (cAMP; in some cases cyclic guanosine monophosphate, cGMP). Effect due to the increase of cyclic adenosine monophosphate is prostaglandin A ₂ depends on the cell type _{(Prostaglandin A 2, PGA 2)} , prostaglandin _{B 2 (Prostaglandin B 2, PGB} 2), prostaglandin C ₂ (Prostaglandin C ₂ , PGC ₂ ) lowers blood pressure, prostaglandin D ₂ (Prostaglandin D ₂ , PGD ₂ ), prostaglandin E ₁ (Prostaglandin E ₁ , PGE ₁ ) is known to inhibit platelet aggregation and participate in inflammatory processes such as pain and fever. Prostaglandin D ₂ is known as a major culprit for aggravating asthma, especially in bronchial asthma patients (Loewen PS, Review of the selective COX-2 inhibitors celecoxib and rofecoxib: focus on clinical aspects.CJEM 4 , pp.268-75 (2002) Ueno A, Inflammation-allergy and prostanoids. (1) .Prostanoids in experimental inflammatory reaction.Nippon Yakurigaku Zasshi 117 , pp.255-61, 2001).

Leukotriene constitutes a group of locally acting hormones produced in vivo from arachidonic acid. Important leukotrienes include leukotriene B ₄ (LTB ₄ ) and leukotriene C _4. (LTC ₄ ), leukotriene D ₄ (LTD ₄ ) and leukotriene E ₄ (LTE ₄ ) Biosynthesis of these leukotrienes begins by the enzyme 5-lipoxygenase acting on arachidonic acid to produce an epoxide known as leukotriene A _4, which is produced by successive enzymatic reaction steps (LTB ₄ , LTC ₄ , LTD ₄ , LTE ₄ ). Leukotriene is an allergic and allergic reaction such as pulmonary artery disease, such as asthma, chronic bronchitis and related obstructive airways disease, upper respiratory tract and allergic rhinitis, contact dermatitis, allergic conjunctivitis, arthritis or inflammatory bowel disease, pain, atopic dermatitis Inflammation and atherosclerosis, including dermatitis (Lotzer K et al, The 5-lipoxygenase pathway in arterial wall biology and atherosclerosis. Biochim Biophys Acta 1736 (1) , pp.30-7, 2005) and heart disease (Back M, Leukotrienes: Potential therapeutic targets in cardiovascular diseases.Bull Acad Natl Med 190 (7) , pp.1511-1518; discussion pp.1518-1521, 2006). Drugs that are recently attracting attention as allergic agents for asthma are drugs that have both histamine free inhibitors, leukotriene C ₄ production inhibitors, and platelet activator inhibitors.

Perennial Inn geumbulcho (Inula belongs to the Asteraceae japonica Thunberg ) is called scallop because only flowers are used as a medicinal herb, and sesquiterpenoid lactone, britanin and inulin are contained in the ground part of the fire. The flower scallop contains a large amount of sterols such as quercetin, isoquercetin, caffeic acid, cholorogenic acid, inulin and taraxasterol. It is known (Information and Shin Min-kyo, by Do Hae Hyang Medicine (Medicinal Medicine), Daelim, Yeonglimsa, pp.1054-1056, 1998).

  Gold fire, which has long been used as a herb, is widely distributed in Korea, China, and Japan (Jiangsu New Medical College.A Dictionary of Tranditional Chinese Drug.Shanghai People's Press: Shanghai, pp.324, 1997; Chinese Academy of Medicine.Institute of Pharmacy Traditional Chinese Medicine, pp 476, 1984).

Although scientific research on the capillary extract has been reported anti-diabetic efficacy (Shan JJ et al, Anti-diabetic and hypolipidemic effects of aqueous-extract from the flower of Inula japonica in alloxan-induced diabetic mice. Biol Pharm Bull 29 , pp. 455-459, 2006), to date, there has been no teaching or description anywhere in the literature regarding the use of compounds isolated from sunning in relation to the therapeutic and prophylactic effects on inflammatory, allergic or asthmatic diseases.

Therefore, the present inventors have found that the compounds isolated in the retrograde are beta-hexosaminidase (b-Hex), COX-2 dependent PGD ₂ LTC ₄ inhibits production and is involved in allergic asthma Inhibition of production was measured to confirm anti-inflammatory and anti-allergic effects.

In order to achieve the above object, the present invention is Tomantosin, Britannin (Britanin) or Quercetagetin (Quercetagetin) 3,4'-dimethyl ether isolated from the extract of the extract as an active ingredient It provides a pharmaceutical composition for the prevention and treatment of inflammatory or allergic diseases.

The present invention is directed to the treatment of inflammatory or allergic diseases containing Tomantosin, Britannin or Quercetagetin 3,4'-dimethyl ether isolated from the extracts of the extract. Provide health functional foods for prevention and improvement.

The extracts defined herein are characterized in that they are linear polar solvent soluble extracts or non-polar solvent soluble extracts.

The polar solvent soluble extract as defined herein comprises a solvent selected from water, methanol, ethanol, butanol or a mixed solvent thereof, preferably water, and an ethanol mixed solvent, more preferably 50 to 80% ethanol mixed solvent. do.

The non-polar solvent soluble extraction compounds as defined herein include extracts soluble in hexane, chloroform, methylene chloride or ethyl acetate, preferably hexane or ethyl acetate.

Inflammatory diseases as defined herein include acute or chronic inflammatory diseases, chronic bronchitis and related obstructive airway diseases, gastritis and rhinitis, arthritis or inflammatory bowel disease, pain, arteriosclerosis, heart disease, multiple sclerosis, Parkinson's disease, Alzheimer's disease, Diseases including colon cancer and the like, preferably, acute or chronic inflammatory diseases, arthritis or inflammatory bowel disease.

Allergic diseases as defined herein include allergic and allergic diseases such as asthma, contact dermatitis, allergic conjunctivitis, allergic reactions, atopic dermatitis, preferably asthma, contact dermatitis or atopic dermatitis.

Hereinafter, the method for obtaining the extract of the present invention will be described in detail.

For example, the scrubbing extract may be washed and dried, and the dried scumber weight is mixed with water, a C ₁ to C ₄ lower alcohol or a mixed solvent thereof, preferably a water and ethanol mixed solvent as an extractant. About 1 to 5 times, preferably 2 to 4 times of, at about 5 to 100 ℃, preferably 20 to 90 ℃, cold extraction, hot water extraction for about 2 hours to 48 hours, preferably 24 hours , Ultrasonic extraction, reflux cooling extraction or heat extraction, preferably extracting by reflux cooling extraction, followed by filtration and concentration under reduced pressure to obtain a crude crude extract of the present invention; Suspension in the crude extract in about 1 to 10 times (w / v), preferably in about 2 to 5 times the volume of water, then about 1 to 10 times (w / v), preferably about A second step of adding a nonpolar solvent such as hexane, ethyl acetate, chloroform, dimethylene chloride, etc. in a volume of 2 to 5 times (w / w) to obtain a nonpolar solvent soluble extractive extract; A third step of fractionating the remaining aqueous solution from the fractionation process with butanol (n-BuOH) or methanol in the same manner as above to obtain a polar solvent soluble extract; Compounds of the present invention may be obtained by subjecting the non-polar solvent soluble extract to silica gel column chromatography, reverse phase column chromatography, or other conventional purification methods well known in the art, and further refining such as recrystallization. have.

The present invention provides a pharmaceutical composition for the prevention and treatment of inflammatory or allergic diseases containing the extract extract compound obtained by the production method as an active ingredient.

The pharmaceutical composition containing the compound of the present invention contains 0.1 to 50% by weight of the extract compound based on the total weight of the composition.

However, the composition is not limited thereto, and may vary depending on the condition of the patient, the type of disease, and the progress of the disease.

Since the compound of the present invention has little toxicity and side effects, it is a drug that can be used safely even when taken for a long time for the purpose of prevention.

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions.

The compositions of the present invention can be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. Carriers, excipients and diluents that may be included in the composition comprising the fractions 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. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid form preparations contain at least one excipient such as starch, calcium carbonate and sucrose in the fraction. Or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. 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.

The preferred dosage of the composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the composition of the present invention is preferably administered at 0.5 g / kg to 5 g / kg, preferably 1 g / kg to 3 g / kg per day. The administration may be carried out once a day or divided into several doses. Accordingly, the dosage is not limited in any way to the scope of the present invention.

The composition of the present invention may be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

The present invention provides a health functional food for the prevention and improvement of inflammatory diseases containing a compound isolated from the extract obtained by the production method as an active ingredient.

The composition comprising the compound of the present invention can be used in various ways, such as drugs, food and beverages for the prevention and improvement of inflammation or allergic diseases.

Examples of the food to which the compound of the present invention may be added include various foods, beverages, gums, teas, vitamin complexes, health supplements, and the like, and may be used in the form of powders, granules, tablets, capsules, or beverages. have.

The amount of the compound in the food or beverage of the present invention may generally be added in an amount of 1 to 5% by weight of the total food weight of the health food composition of the present invention, and the health beverage composition may be 0.02 to 10 g based on 100 ml, preferably Can be added in a ratio of 0.3 to 1 g.

The health beverage composition of the present invention, in addition to containing the above compound as an essential ingredient in the indicated ratio, there is no particular limitation on the liquid component, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. have. Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like Sugar, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of said natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 mL of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the compositions of the present invention may contain flesh for the production of natural fruit juices and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

The composition of the present invention containing a compound isolated from the extract of the present invention as an active ingredient is cyclooxygenase-2 (COX-2) and lipoxygenase (5-LO) that causes various inflammation and allergic diseases It inhibits the activity of eicosanoids and prevents the release of histamine from mast cells, which is useful as a medicine or health functional food for the prevention and treatment of various inflammatory or allergic diseases. Can be used.

 1 is a diagram illustrating a separation process of separating compounds from the ectopic extract.

Hereinafter, the present invention will be described in detail by reference examples, examples and experimental examples.

However, the following reference, Examples and Experimental Examples are merely to illustrate the present invention, the contents of the present invention is not limited to the following Reference Examples, Examples and Experimental Examples.

Example  One. Conquest  Extraction and Separation of Active Ingredients

1.1. Crude extract  Produce

Dry extract (Omni Herbb Co., Ltd., www.omniherb.com ) After repeated extraction for 3 hours with refrigerated reflux extraction at about 60 ℃ for 24 hours with 10L of 15% 70% ethanol (EtOH), each extract The filtrate was filtered through a filter paper, the filtrates were combined, concentrated under reduced pressure with a reduced pressure concentrator (Eyela, Tokyo Rikakikai CO., Tokyo, Japan), lyophilized with a freeze dryer (Labconco, Kansas City, MO, USA), and dried ethanol extract 1.55 kg was obtained.

1.2. Fractions  Produce

After dissolving in 1.5L of distilled water in the ethanol extract prepared in step 1-1, 12L of hexane ( n- Hexane) was added to the fraction to obtain 159g of the hexane soluble fraction.

The remaining aqueous solution from the above experiment was fractionated in the same manner as above to obtain 12L of ethyl acetate, and 8L of butanol to obtain ethyl acetate soluble fraction (EtOAc, 309g), and butanol soluble fraction ( n- BuOH, 361g), respectively. The remaining water-soluble fraction (510 g) was lyophilized with a lyophilizer (Labconco) to obtain fractions.

1.3 Purification and Separation of Compounds

         100 g of the ethyl acetate fraction obtained in step 1-2 were subjected to normal phase column chromatography. First, fill the column (120 × 600 mm) with silica gel (No.9385, 230-400 mesh, Merck) about 30cm and elution with 3L of hexane to make the stationary phase homogeneous, and then ethyl acetate fraction. 100 g was adsorbed onto 250 g of silica gel (No.7734, 70-230 mesh, Merck) and loaded onto the column. Thereafter, 27 small fractions (fraction; In-1 to 27) were obtained by distilling the polarity of the developing solvent in order to sequentially increase the polarity of hexane-ethyl acetate and ethyl acetate-methanol (MeOH).

Tomantosin ( 1 ) (2 g, hereinafter referred to as “SBH-1”) was obtained from the subfraction In-10. In-13 in the fractions was recrystallized with 100% chloroform to obtain Britannin (Britanin; 2 ) (5 g, hereinafter referred to as "SBH-2"). The fraction In-24 was obtained by using a reverse-phase column (4 × 50 cm, LiChroprep RP-18) using a developing solvent (methanol: H ₂ O = 10: 90 to 100% methanol). Quercetagetin 3,4'-dimethyl ether, using column (waters COSMOSIL 5C-18-AR; 5 μm, (250 mm × 20 mm) column) with preparative HPLC (dimethyl ether; 3 ) (38 mg, hereinafter referred to as “SBH-3”) was obtained (FIG. 1).

delete

Reference Example  1. Preparation of experimental materials

PGD ₂ , LTC ₄ Enzyme linked immunoassay (EIA) Kit, N- [2- (cyclohexyloxy) -4-nitrophenyl] -methanesulfonamide (NS398) was purchased from Cayman Chemical (Ann Arbor, MI, USA) and used as a culture medium. RPMI-1640, Modified Eagle Medium (MEM) non-essential amino acids solution, penicillin-streptomycin is GIBCO-BRL (Carlsbad, CA, USA), fetal bovine serum (FBS) is Hyclone (Logan, UT , USA) was used.

Reference Example  2. Cell Culture

Mouse bone marrow-derived mast cells (BMMC) were isolated from bone marrow from male BALB / C mice (Dae Biolink, 6 weeks old, 23 grams, Eumseong, Korea), 10% FBS, 100U / 90% cultured for about 3 weeks in RPMI-1640 medium containing ml penicillin, 100µg / ml streptomycin and IL-3 (the supernatant obtained by stimulating pokeweed mitogen to mouse spleen cells to the final 20%) The above homogeneous BMMC was obtained.

Experimental Example  1. Cell Viability Confirmation Experiment

In order to confirm the BMMC survival rate obtained in the above example, the experiments described in the literature were applied as follows (Jin M et al, The naturally occurring flavolignan, deoxypodophyllotoxin, inhibits lipopolysaccharide-induced iNOS expression through the NF-kappaB activation in RAW264.7 macrophage cells.Biol Pharm Bull 31 (7) , pp. 1312-15, 2008).

1Ｘ10 ⁶ cells / ml BMMC was treated with SBH-1, SBH-2, and SBH-3 at different concentrations, and preincubated at 37 ° C and 5% CO ₂ for 4 hours, followed by 0.5 mg / ml MTT concentration. After treatment for 4 hours, 0.04N of HCI / isopropanol was added, the cells were completely lysed, and the absorbance was measured at 540 nm using an ELISA to calculate the survival rate. As a result, there was no cytotoxicity even at the final concentration of 25 μM (survival rates of SBH-1, SBH-2 and SBH-3 were 98%, 94% and 100%, respectively).

Experimental Example  2. Beta - hexosaminidase   (β- Hex Experiment of inhibitory effect on release

In order to confirm the degree of degranulation by measuring beta-hexosaminidase (β-hex) enzyme activity, which is a degranulation marker of the obtained BMMC of the above example, the experiment described in the literature was applied as follows (Son JK et al, Ginkgetin, a Biflavone from Ginko biloba leaves, inhibits cyclooxygenases-2 and 5-lipoxygenase in mouse bone marrow-derived mast cells.Biol Pharm Bull 28 (12) , pp.2181-84, 2005).

SBH-1, SBH-2 and SBH-3 were treated in BMMC of 2Ｘ10 ⁵ cells / well by concentration to 37 ℃, 5% CO ₂ After incubation for 30 minutes under the conditions, it was stimulated with KL (100 ng / ml) (c-kit ligand, STEMCELL Technologies, Vancouver, Canada), incubated for 15 minutes, and centrifuged for 5 minutes at 3000 rpm and 4 ° C. The supernatant was mixed with β-hex substrate [100 mM citrate buffer (citric acid 0.955%, sodium citrate dihydrate 1.478%, pH 4.5), 1.3 mg / ml p -nitrophenyl-N-acetyl-bD-glucosaminide] and 1: 2 (w / v). After mixing at 37 ° C. for 1 hour and stopping the reaction with 0.2M glycine (pH 10.7) (Sigma), using a ELISA reader (Tecan System, San Jose, CA, USA) Absorbance was measured at, and the measured value was converted into the release rate (release%).

As a result of the experiment, SBH-2 and SBH-3 inhibited 97.6% and 94.7% at 25 mM concentration, respectively, and IC ₅₀ for β-hex was 5.1 μM and 1.2 μM, respectively (Table 1).

Experimental Example  3. COX -2 dependent PGD ₂  Generation inhibition measurement

In order to confirm the degree of inhibition against allergic diseases of the substances isolated in the above example, the production amount of prostaglandin D ₂ (PGD ₂ ) was measured as follows (Son JK et al, Ginkgetin, a Biflavone from Ginko biloba leaves, inhibits cyclooxygenases-2 and 5-lipoxygenase in mouse bone marrow-derived mast cells.Biol Pharm Bull 28 (12) , pp.2181-84, 2005).

After 3 weeks of BMMC culture, the isolated material obtained in Example was treated for 30 min at a cell concentration of 2Ｘ10 ⁵ cells, followed by 100ng / ml KL 100ng / ml LPS (Lipopolysaccharde, Sigma) and 100 U / ml IL-10 After treatment with mixed stimulant (Sigma), the supernatant PGD ₂ was transferred to EIA using PGD ₂ assay kit (Cayman PGD ₂ EIA kit PGD ₂ -MOX EIA kit, product no. 512011) after 8 hours of cell stimulation. Measured.

At this time, in order to suppress the production of PGD ₂ produced by COX-1 (cyclooxygenase-1), 10 μg / ml of aspirin was pretreated with BMMC 1 hour beforehand.

As a result of the above experiment, SBH-1, SBH-2 and SBH-3 are 96.6%, respectively, the PGD ₂ produced at the concentration of 25μM, were inhibited 98.5%, 95%, PGD ₂ production of SBH-1 and SBH-4 The IC _{50 for} was 13.3 μM and 6.5 μM, respectively (Table 1).

Experimental Example  4. Leukotrien  Impact Analysis Experiments on Generation

In order to examine the effect on the LTC ₄ production of the separated substances in the above example was tested by applying the method disclosed in the literature (Yang JH et al, Anti-allergic activity of an ethanol extract from Salviae miltiorrhiza . Arch Pharm Res 31 (12) , pp. 1597-603, 2008).

After 3 weeks of BMMC culture, mast cells were pretreated with the separated substances obtained in Example for 30 minutes in advance, and then treated with SCF stimulant (Sigma, S9915) for 15 minutes. LTC ₄ Determination of cell supernatants after stimulation was measured by _{LTC 4 EIA (Enzyme linked immuno assay} , Cayman Inc., product no. 520211) kit.

As a result of the above experiment, SBH-1, SBH-2 and-3 SBH is LTC ₄ at a concentration of 25μM Production was inhibited by 23.1%, 99.1% and 99.9%, respectively, and LTC ₄ of SBH-2 and SBH- ₄ IC ₅₀ for generation was 3.1 μM and 0.3 μM, respectively (Table 1).

matter Inhibition (%) IC ₅₀ PGD ₂ LTC ₄ β-Hex PGD ₂ LTC ₄ β-Hex 25 μM 25 μM 25 μM SBH-1 96.9 23.1 24.7 13.3 μM - - SBH-2 98.5 99.1 97.6 - 3.1 μM 5.1μM SBH-3 95.0 99.9 94.7 6.5 μM 0.3 μM 1.2 μM Positive control - - - NS398 AA861 DPT 0.1 nM 2.4 nM 27.5 nM

Examples of the formulation of the composition comprising the compound of the present invention will be described above, but the present invention is not intended to be limited thereto, but is intended to be described in detail.

Formulation example  One. Powder  Produce

       SBH-1 20 mg

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

Formulation example  2. Preparation of tablets

       SBH-1 10 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components and tableting according to the conventional tablet manufacturing method to prepare a tablet.

Formulation example  3. Preparation of capsules

       SBH-1 10 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

Formulation example  4. 1st injection

      SBH-1 10 mg

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na2HPO4,12H2O 26 mg

(2 ml) per 1 ampoule according to the usual injection preparation method.

Formulation example  5. Liquid  Produce

      SBH-1 20 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

Each component was added to purified water in accordance with the usual liquid preparation method and dissolved, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was adjusted to 100 ml with purified water, And sterilized to prepare a liquid preparation.

Formulation example  6. Manufacture of health food

       SBH-1 1000 mg

Vitamin mixture quantity

70 [mu] g of vitamin A acetate

Vitamin E 1.0 mg

0.13 mg vitamin B1

0.15 mg of vitamin B2

0.5 mg vitamin B6

0.2 [mu] g vitamin B12

10 mg vitamin C

Biotin 10 μg

Nicotinic acid amide 1.7 mg

50 ㎍ of folic acid

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

15 mg of potassium phosphate monobasic

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

100 mg of calcium carbonate

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified. The granules may be prepared and used for preparing a health food composition according to a conventional method.

Formulation example  7. Manufacture of health drinks

      SBH-1 1000 mg

Citric acid 1000 mg

100 g of oligosaccharide

Plum concentrate 2 g

Taurine 1 g

Purified water was added to a total of 900 ml

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was heated at 85 DEG C for about 1 hour with stirring, and the solution thus prepared was filtered to obtain a sterilized 2-liter container, which was sealed and sterilized, ≪ / RTI >

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

Claims (8)

Acute or chronic inflammatory disease, chronic bronchitis, containing Tomantosin, Britannin or Quercetagetin 3,4'-dimethyl ether isolated from the extracts Pharmaceutical composition for the prevention and treatment of upper respiratory tract, rhinitis, arthritis, inflammatory bowel disease, asthma, contact dermatitis, allergic conjunctivitis, or atopic dermatitis. The method of claim 1,
The extract is a pharmaceutical composition that is a polar solvent soluble extract or a non-polar solvent soluble extract The method of claim 2,
The polar solvent soluble extract is a pharmaceutical composition is a solvent selected from water, methanol, ethanol, butanol or a mixed solvent thereof. The method of claim 2,
Wherein said nonpolar solvent-soluble extracting compound is a solvent selected from hexane, chloroform, methylene chloride or ethyl acetate solvent. delete delete Acute or chronic inflammatory disease, chronic bronchitis, containing Tomantosin, Britannin or Quercetagetin 3,4'-dimethyl ether isolated from the extracts Health functional foods for the prevention and improvement of upper respiratory tract, rhinitis, arthritis, inflammatory bowel disease, asthma, contact dermatitis, allergic conjunctivitis, or atopic dermatitis. A dietary supplement according to claim 7 which is a powder, granule, tablet, capsule or beverage.

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