KR101715274B1 - Composition containing extract or fractions of Chrysanthemum indicum L. for treating, improving or preventing inflammatory disease - Google Patents

Abstract

(COX-2), tumor necrosis factor (TNF-alpha), and myelin (TNF-alpha). The present invention relates to a composition for preventing, improving or treating an inflammatory disease, It is possible to reduce the concentration of peroxidase (MPO), reduce the expression amount of nitrogen monoxide, iNOS, and have excellent anti-inflammatory activity through inhibition of nuclear factor-kB (NF-kB) And a composition for preventing, ameliorating or treating an inflammatory disease, which comprises an extract or fraction thereof as an active ingredient.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for preventing, improving or treating an inflammatory disease, which comprises an extract of Chrysanthemum morifolium or a fraction thereof as an active ingredient.

The present invention relates to a composition for preventing, improving or treating an inflammatory disease, and more particularly, to a composition for preventing, ameliorating or treating an inflammatory disease comprising an extract or a fraction thereof as an active ingredient.

Inflammation is one of the defense mechanisms to prevent and repair further damage when the tissue of our body is damaged. Although we will focus primarily on the response to microbial infections here, there are many other causes besides microbial infections that cause the inflammation. For example, an inflammatory response can be caused by damage caused by compounds that are stung, nicked, or nicked by something that is burned or sharp. Symptoms of inflammation include reddened, inflamed, inflamed, inflamed (inflamed) and inflamed (inflamed) areas, as Celsus, a Roman physician described well. There is also a loss of functionality here. When the cause of inflammation is soon removed by inflammation, the inflammation disappears. The inflammation that occurs in a relatively short period of time is called acute inflammation. However, if the cause of inflammation is not removed in a short period, it can last for several weeks and sometimes for a long period of years. Such inflammation is called chronic inflammation. The function of the inflammatory reaction is to remove the cause of the injury (such as the toxin produced by the infecting organism or microorganism) from our body, to limit it to a narrow area so that damage can not be widespread if it can not be completely removed from our body And to restore and restore the damaged area.

Damage to certain tissues usually results in capillary damage, and blood platelets are exposed to extracellular components, resulting in platelet aggregation. Serotonin, which is stored in the platelets, is released. Serotonin causes other platelets to circulate around and contracts the blood vessels. Also, broken blood vessels can be easily clogged by the coagulating blood, which quickly prevents further loss of blood. Mast cells in the skin or mucous membranes are damaged as a result of damage to tissues. Histamine, which is already formed in the cells, may leak, and mast cells may be stimulated by the complement proteins when the complement system is activated Histamine may also be released. Kinins play an important role in the chemotaxis of granule cells (especially neutrophils) when they are activated by plasma proteins. Prostaglandins are also released by damaged cells, further enhancing the function of histamine or kinin proteins. Mast cells and basophils also release leukotrienes, which also serve to dilate blood vessels and attract phagocytes. When phagocytes ingest and activate microorganisms, they secrete various cytokines to attract other devices of the immune system Activate.

Thus, when the tissue is damaged in our body, a variety of mechanisms detect the situation of our body and generate a signal. In a particular situation, which mechanism will work first and the main mechanism will depend on the specific conditions of the situation. Depending on the situation, the damage can be detected first by the mast cells, and the infected microorganisms immediately after the damage can be detected by the phagocytes next to them, and other mechanisms can be activated. However, it is a consistent strategy to address these various mechanisms, which is to expand the vessels in the damaged area and increase the permeability of the blood vessels. The expansion of the blood vessels causes the blood to multiply in the injured area, and it has the effect of attracting plasma components and nuclides required for the inflammatory reaction to the damaged area. It also serves to dilute harmful substances in the injured area. Histamine increases the permeability of blood vessels, allowing plasma to escape from the blood vessels to surrounding tissues and allowing leukocytes to escape from the blood vessels and into damaged tissues. The body fluids from the blood vessels wash the damaged areas and carry harmful substances into the lymphatic vessels and transport them to the nearby lymph nodes. In the lymph nodes, many immune cells (macrophages, lymphocytes) are concentrated, and adaptive immune responses are initiated. The phenomenon that the liquid component of the blood flows into the tissue and the tissue becomes taut is called edema. In addition, the blood is drawn to the wound area and red, and heat is generated by the active flow of blood. Edema also makes the nerves feel pain.

The inflammatory response at the site of infection is initiated by the response of macrophages to the pathogen. Inflammatory mediators such as reactive oxygen species and active nitrogen species, prostaglandins, leukotrienes, and inflammatory cytokines such as TNF-α, IL-6 and IL-8 produced by macrophages activated by pathogenic agents, (Renauld JC, New insights into the role of cytokines in asthma, J. Clin. Pathol. 54, pp. 577-589, 2001; Blake GJ, Ridker PM, Tumor necrosis factor-α, inflammatory biomarkers , and atherogenesis. Eur. Heart J. 23, pp.345347, 2002). Cytokines are produced by various cells and have a variety of actions. It is difficult to classify cytokines into specific classes, because even if they are the same cytokines, their actions vary. T cells are recognized by macrophage-mediated antigens and are activated by a number of cytokines when they induce an immune response. Activation of NF-κB, a transcription factor of genes involved in the production of these inflammatory mediators, is crucial to the inflammatory action of macrophages. Inflammatory genes such as inducible nitric oxide synthase (iNOS), cyclooxygenase (COX-2), TNF-α, IL-6 and IL- lt; / RTI >

Non-steroidal anti-inflammatory drugs (NSAIDs) are the most potent anti-inflammatory drugs developed by humans so far. Nonsteroidal anti-inflammatory drugs are analgesic, antipyretic and anti-inflammatory drugs. However, when the non-steroidal anti-inflammatory agent is used for a long period of time, side effects such as stomach abdominal pain, stomach and duodenal ulcer, diarrhea, pancreatic dysfunction, gastrointestinal bleeding, hepatotoxicity, renal dysfunction, platelet aggregation inhibition, headache, tinnitus, dizziness, Accompanied by.

On the other hand, the munguk has been drinking tea as a perennial herb belonging to the Compositae and has been used in oriental medicine for dizziness, fever, inflammation, hypertension and respiratory diseases. Studies on the physiological activity of the germplasm have been reported to have antimicrobial activity, antiviral activity, antioxidant activity and immunoregulatory activity. Various components such as flavonoid compounds, terpenoid compounds, and phenolic compounds have been reported as main components of germ-germ cells. As described above, various pharmacological effects of Ganoderma lucidum are known, but studies on the effect of extracts and fractions of Ganoderma lucidum on the diseases are insufficient.

Korean Patent Laid-Open No. 10-2011-0081435 Korean Patent Laid-Open No. 10-2012-0021494

In order to solve the problems of the prior art as described above, the present invention relates to a method for reducing COX-2, tumor necrosis factor (TNF-a) and myeloperidase (MPO) It is an object of the present invention to provide a composition for preventing, ameliorating or treating an inflammatory disease which can reduce the expression amount of enzyme (iNOS).

It is another object of the present invention to provide a composition for preventing, ameliorating or treating an inflammatory disease which shows excellent anti-inflammatory effects through inhibition of nuclear factor-kB (NF-kB) activity.

In order to accomplish the above object, the present invention provides a pharmaceutical composition for preventing or treating an inflammatory disease, which comprises an extract or fraction thereof as an active ingredient.

(B) adding distilled water to the extract, and adding the extract to the extract; and (c) adding the distilled water to the extract to obtain an extract from the extract, wherein the extract or fraction is obtained by extracting the extract with a solvent selected from water, an alcohol having 1 to 4 carbon atoms and a mixed solvent thereof, And sequentially fractionating them using a solvent to obtain respective solvent fractions.

In particular, the alcohol having 1 to 4 carbon atoms in step (a) is preferably selected from methanol, ethanol, isopropanol and butanol, and the organic solvent in step (b) is selected from among dichloromethane, ethyl acetate and water It is preferable that at least one is.

The gut fractions can be obtained by using linarin, acacetin-7-ObD-rutinoside, tricin, N- [tris (hydroxymethyl) methyl] glycine, isorhamnetin 3-O-? -D-glucopyranoside D-glucopyranoside, apigenin-7-O-beta-D-glucopyranoside, methyl 3,5-di-glucopyranoside, OE-caffeoyl-quinate, quercetin, quercetin-7-O-beta-D-glucopyranoside, -D-glucopyranoside, 3-beta-D-glucopyranosyl-3-epi-2-isocucuric acid, Glucosidase inhibitors such as pinoresinol-4-O- beta -D-glucopyranoside ((+) - pinoresinol-4-0B-D-glucopyranoside), cyclingresinol- (-) - cis-chrysanthenol-β-D-glucopyranoside, (-) - cis-chrysanthenol-β-D-glucopyranoside, A compound represented by the formula (2) It may be at least any one selected from the compounds.

[Chemical Formula 1]

(2)

The cuttlefish extract or fraction is preferably contained in an amount of 0.01 to 95% by weight based on the total weight of the composition.

The hamster extract or fraction may reduce the concentration of cyclooxygenase (COX-2), tumor necrosis factor (TNF-a) or myeloperoxidase (MPO).

The hamster extract or fraction may reduce the expression amount of nitrogen monoxide (NO) or inducible nitric oxide synthase (iNOS).

In addition, the hamster extract or fraction can inhibit nuclear factor kappa B (NF-kB) activity and inhibit inflammation in macrophages.

The present invention also provides a food composition for preventing or ameliorating an inflammatory disease, which contains an extract or fraction thereof as an active ingredient.

The cuttlefish extract or fraction is preferably contained in an amount of 0.01 to 95% by weight based on the total weight of the composition.

In accordance with the present invention, there is provided a method of decreasing the levels of cyclooxygenase (COX-2), tumor necrosis factor (TNF-a) and myeloperidase (MPO), and reducing the expression amount of nitric oxide and nitric oxide synthase Inflammatory effect through the inhibition of nuclear factor-kB (NF-kB) activity, and it has excellent antiinflammatory effect through inhibition of nuclear factor-kB (NF-kB) activity and can be used for the treatment of gastritis, gastric ulcer, bronchitis, asthma, edema, hepatitis, nephritis, , Rheumatoid arthritis, osteoarthritis, joint inflammation related diseases, cancer, degenerative diseases and the like.

FIG. 1 is a schematic diagram showing a method for separating Momordicae radix extract, fraction and 13 single fractions according to an embodiment of the present invention.
FIG. 2 is a graph showing the effect of extracts and fractions on the cell viability according to an embodiment of the present invention. FIG.
FIG. 3 is a graph showing the effect of thirteen varieties of germinated single fractions on cell viability according to an embodiment of the present invention.
FIG. 4 is a graph showing the expression levels of nitrogen monoxide (NO) in RAW 264.7 cells in the treatment of hamster extract and fraction according to an embodiment of the present invention.
FIG. 5 is a graph showing the expression levels of carbon monoxide (NO) in RAW 264.7 cells in the treatment of thirteen variegated single fractions according to an embodiment of the present invention.
FIG. 6 is a graph showing the expression levels of tumor necrosis factor (TNF-a) in RAW 264.7 cells in the treatment of hamster extract and fraction according to an embodiment of the present invention.
FIG. 7 is a graph showing the expression levels of tumor necrosis factor (TNF-α) in RAW 264.7 cells in the treatment of thirteen locomotor single fractions according to an embodiment of the present invention.
FIG. 8 is a graph showing the expression levels of cyclooxygenase-2 (COX-2) and iNOS in RAW 264.7 cells treated with Ganoderma lucidum ethanol extract according to an embodiment of the present invention.
FIG. 9 is a graph showing the expression levels of cyclooxygenase-2 (COX-2) and iNOS in RAW 264.7 cells in the treatment of acetic acid ethyl acetate fraction according to an embodiment of the present invention.
Figure 10 is a block diagram of an embodiment of the present invention (COX-2) and iNOS expression in RAW 264.7 cells in the treatment of GM whole water fraction.
FIG. 11 is a graph showing an inflammation-inhibiting effect in an inflammatory bowel disease model in the treatment of ganoderma ethanol extract according to an embodiment of the present invention. Fig. 11A shows the change in body weight, Fig. 11B shows the colon length, Fig. 11C shows the DAI score, Fig. 11D shows the MPO activity, and Fig.
FIG. 12 is a graph showing an inflammation-suppressing effect in an inflammatory bowel disease model in the case of treatment of a water-extract of Korean red ginseng root according to an embodiment of the present invention. Fig. 12A shows the change in body weight, Fig. 12B shows the colon length, Fig. 12C shows the DAI score, and Fig. 12D shows the MPO activity results.
FIG. 13 is a graph showing an inflammation-inhibiting effect in an inflammatory bowel disease model in the treatment of acetic acid ethyl acetate fraction according to an embodiment of the present invention. Fig. 13A shows the change in body weight, Fig. 13B shows the colon length, Fig. 13C shows the DAI score, Fig. 13D shows the MPO activity, and Fig. 13E shows the Weston blot analysis result.
FIG. 14 is a graph showing an inflammation-inhibiting effect in an inflammatory bowel disease model in the case of treatment of the red ginseng water fraction according to an embodiment of the present invention. Fig. 14A shows the change in body weight, Fig. 14B shows the colon length, Fig. 14C shows the DAI score, Fig. 14D shows the MPO activity, and Fig. 14E shows the Weston blot analysis result.

Hereinafter, the present invention will be described in detail.

The present inventors have conducted studies on extracts and fractions thereof to develop new drugs. As a result, it has been confirmed that the extracts or fractions of the extract have an excellent anti-inflammatory effect against inflammation including colitis. Especially, And thus the present invention has been completed on the basis thereof.

The composition for preventing or treating an inflammatory disease according to the present invention is characterized by comprising an extract or fraction thereof as an active ingredient.

The extract or fraction of Chrysanthemum indicum L. can be extracted from various parts of the cuttlefish, preferably extracted from flowers, leaves, stems, roots or outposts of cuttlefish, more preferably from flowers or leaves of cuttlefish , And most preferably from a flower of a cuttlefish.

The cuttlefish extract or fraction can be obtained by extracting, isolating and fractionating from the nature using a method of extraction, separation and fractionation known in the art. The 'extract' defined in the present invention is obtained by extraction treatment from red ginseng using an appropriate solvent and includes, for example, crude extract, polar solvent-soluble extract or non-polar solvent-soluble extract of red ginseng.

The extract can be extracted according to various extraction solvents and extraction methods, and any suitable solvent for extracting the extract or fractions from the extract can be any pharmaceutically acceptable organic solvent, and water or an organic solvent But is not limited thereto. Examples of the solvent include water, alcohols having 1 to 4 carbon atoms such as methanol, ethanol, propanol, isopropanol, and butanol, etc., Can be mixed and used. Particularly, as the solvent, methanol or ethanol (alcohol) is preferably used, and ethanol (alcohol) is particularly preferably used.

The extraction temperature is preferably 50 to 100 ° C. As the extraction method, methods such as hot water extraction method, cold extraction method, reflux cooling extraction method, solvent extraction method, steam distillation method, ultrasonic extraction method, elution method and compression method can be used and more preferred is the use of hot water extraction method or reflux cooling extraction method Do.

In addition, the gum-like fractions can be obtained by fractionating the organic extract from non-polar to polar fractions of the gum extract obtained as described above.

Suitable fractions for fractionation of the gut fractions can be water, ethanol, methanol, hexane, chloroform, dichloromethane, ethyl acetate, butanol or mixtures thereof. Particularly, dichloromethane, ethyl acetate and water And a fraction obtained by fractionation. Particularly, the water fraction and the ethyl acetate fraction are remarkably excellent in anti-inflammatory activity as compared with the other fractions. Therefore, the water-reducing fraction of the present invention is more preferably a water fraction, an ethyl acetate fraction or a mixture thereof.

After the extract is obtained using water or an organic solvent as described above, a liquid material can be obtained by freezing, heating and filtering at room temperature by a conventional method known in the art, or a liquid material can be obtained by further evaporating, spray drying or freeze- You may.

In addition, the gum extract or fraction contained as an active ingredient in the composition of the present invention may be prepared in powder form by an additional process such as vacuum distillation, freeze-drying, spray-drying, and the like, after extracting and fractionating the extract or fraction as described above. The extract or fraction may be further purified by further chromatography using various chromatographies such as silica gel column chromatography, thin layer chromatography, high performance liquid chromatography and the like Can be obtained.

Therefore, the cuttlefish extract and fractions to be used in the present invention include all the extracts, fractions and tablets obtained in each step of extraction, fractionation or purification, their diluted solutions, concentrates or dried products.

The extract or fraction of the extract of the present invention, which has been extracted or fractionated as described above, has excellent antiinflammatory effects in a mouse model inducing ulcerative colitis and is also excellent in a cell model that induces inflammation using LPS (lipopolysaccharide) in RAW 264.7 cells It has an anti-inflammatory effect. In particular, thirteen monocomponent components of the hamsters extract or fraction, which is an active ingredient of the present invention, show a superior anti-inflammatory effect.

Wherein the thirteen monocomponents are selected from the group consisting of linarin (acacetin-7-ObD-rutinoside, tricin, N- [tris (hydroxymethyl) methyl] glycine, O-β-D-glucopyranoside, apigenin-7-O-β-D-glucopyranoside, β-D-glucopyranoside, methyl 3,5-di-OE-caffeoyl-quinate, quercetin, quercetin, 8), quercetin-7-O-? -D-glucopyranoside (9), 3-beta-D-glucopyranosyl- 3-epi-2-isocucuric acid, (+) - pinoresinol-4-O- beta -D-glucopyranosyl-3-epi-2- D-glucopyranoside, (+) - pinoresinol-4-O- beta -D-glucopyranoside, the following formula (11), syringaresinol-O-beta-D-glucopyranoside e, represented by the following formula (12), (-) - cis-chrysanthene-β-D-glucopyranoside ((-) - cis-chrysanthenol- And a compound represented by the following formula (2).

[Chemical Formula 1]

(2)

(3)

[Chemical Formula 4]

[Chemical Formula 5]

[Chemical Formula 6]

(7)

[Chemical Formula 8]

[Chemical Formula 9]

[Chemical formula 10]

(11)

[Chemical Formula 12]

[Chemical Formula 13]

The extracts or fractions of the extract of the present invention which are effective components of the present invention, particularly the above-mentioned 13 kinds of extracts or fractions of the extract of the present invention, can be used for the treatment of various diseases such as cyclooxygenase (COX-2), tumor necrosis factor (TNF-α) and myeloperidase (nuclear factor kappa B, NF-kB) activity by reducing the concentration of nitric oxide synthase (iNOS) and reducing the concentration of nitric oxide synthase (iNOS) And inhibit inflammation in macrophages.

Therefore, in the present invention, lipopolysaccharide (LPS) is treated with RAW 264.7 cells to increase the expression of mediating proteins involved in inflammation, and the amount of nitric oxide (NO) synthesized in the cells is measured Thus, it was confirmed that the intestinal extract or fraction, which is an active ingredient of the present invention, decreased intracellular nitrogen monoxide synthesis inhibitory rate in a concentration-dependent manner, and inhibited the intracellular synthesis of TNF-α, which is an index of inflammatory response.

Therefore, the extract or fraction thereof of the present invention can be used as a medicine or a health functional food useful for prevention, treatment or improvement of an inflammatory disease.

The inflammatory disease refers to a disease caused by a common inflammatory reaction and specifically includes gastritis, gastric ulcer, bronchitis, asthma, edema, hepatitis, nephritis, arteriosclerosis, inflammatory bowel disease (IBD) Rheumatoid arthritis, osteoarthritis, joint inflammation related diseases, cancer, degenerative diseases, and the like.

Accordingly, the present invention provides a pharmaceutical composition for preventing or treating an inflammatory disease comprising an extract or fraction thereof as an active ingredient.

The gut extract or fraction is preferably contained in an amount of 0.01 to 95% by weight, more preferably 1 to 80% by weight based on the total weight of the composition. If the content is less than 0.01% by weight, the efficiency of taking may be poor. If the content is more than 95% by weight, formulation may be difficult.

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions. In addition, it can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, oral preparations such as syrups and aerosols, external preparations, suppositories and sterilized injection solutions according to a conventional method. Suitable formulations known in the art are preferably those as disclosed in Remington ' s Pharmaceutical Science, recently, Mack Publishing Company, Easton PA. Examples of carriers, excipients and diluents which may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like. When the composition is formulated, it is prepared using a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient usually 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, lactose, Gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and 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 sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. 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 butter, laurin, glycerogelatin and the like.

The term "administering" as used herein is meant to provide any desired composition of the invention to a subject in any suitable manner.

The present invention relates to pharmaceutical compositions comprising an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as contemplated by a researcher, veterinarian, physician or other clinician, RTI ID = 0.0 > effective < / RTI > amount. It will be apparent to those skilled in the art that the therapeutically effective dose and frequency of administration for the pharmaceutical compositions of the present invention will vary with the desired effect. Thus, the optimal dosage to be administered can be readily determined by those skilled in the art and will vary with the nature of the disease, the severity of the disease, the amount of active and other ingredients contained in the composition, the type of formulation, and the age, The age, body weight, sex, diet, time of administration, route of administration and fraction of the composition, duration of treatment, concurrent medication, and the like. For a desired effect, the pharmaceutical composition of the present invention may be administered in an amount of 1 to 10,000 mg / kg / day, preferably 1 to 200 mg / kg / day, or may be administered once a day, It may be administered separately.

The pharmaceutical compositions of the present invention can be administered to a subject in a variety of routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection.

The composition of the present invention can also be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy or biological response modifiers for cancer prevention or treatment.

The present invention also provides a food composition for preventing or ameliorating an inflammatory disease, which contains an extract or fraction thereof as an active ingredient. When the cuttlefish extract or fraction of the present invention is used as a food additive, the cuttlefish extract or fraction may be added as it is, mixed with other food or food ingredients, and used appropriately according to a conventional method.

In addition, it is of course possible to suitably change the mixing amount of the extract or fraction thereof, which is an effective ingredient, according to the purpose of use (prevention, health or therapeutic treatment), and the extract or fraction of the extract is in the range of 0.01 to 95 By weight, and more preferably from 1 to 80% by weight. If the content is less than 0.01% by weight, the efficiency of taking may be poor. If the content is more than 95% by weight, formulation may be difficult.

As a specific example, at the time of manufacturing a food or beverage, the cuttlefish extract or fraction of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less, based on the raw material. However, when it is intended for health and hygiene purposes or for the purpose of controlling health, it can be added in an amount below the above range, and there is no problem in terms of safety. Therefore, the active ingredient can be used in an amount exceeding the above range have.

There is no particular limitation on the kind of the food. Examples of the food to which the hamster extract or fraction of the present invention can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, , Tea, a drink, an alcoholic beverage, a vitamin complex, and the like.

When the food composition of the present invention is prepared as a beverage, it may contain additional ingredients such as various flavors or natural carbohydrates such as ordinary beverages. Examples of the natural carbohydrate include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; Natural sweeteners such as dextrin and cyclodextrin, synthetic sweeteners such as saccharin and aspartame, and the like. The natural carbohydrate is contained in an amount of 0.01 to 10% by weight, preferably 0.01 to 0.1% by weight based on the total weight of the food composition of the present invention.

In addition to the above, the food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, , Carbonating agents used in carbonated beverages, and the like. In addition, the compositions of the present invention may include flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of the above additives is not limited to a great extent, but may be in the range of 0.01 to 0.1% by weight based on the total weight of the food composition of the present invention.

Hereinafter, the present invention will be described in more detail with reference to examples. These embodiments are for purposes of illustration only and are not intended to limit the scope of protection of the present invention.

Example 1 Preparation of Tumor Root Extract

3.0 ㎏ of Chrysanthemum indicum flowers (CI), which was dried in the air, was extracted three times with 70% ethanol using a reflux extracting machine to obtain 500 g (yield: 16.67%) of Chum koji extract.

Example 2. Preparation of gut fractions

500 g of the mugwort extract obtained in Example 1 was dissolved in water and then the dichloromethane (CH ₂ Cl ₂ ) layer was separated three times. The dichloromethane layers thus obtained were combined and concentrated under reduced pressure to obtain 200 g (yield: 40%) of a powdery tungstoc dichloromethane fraction. To the remaining water layer was added ethyl acetate (CH ₃ COOC ₂ H ₅ ) and fractionation was carried out in the same manner as above. The filtrate was concentrated under reduced pressure and lyophilized to obtain 110 g (yield: 22%) of powdery gum tragacanth ethyl acetate fraction and 190 g 38%).

Example 3. Isolation of 13 single fractions

As shown in FIG. 1, a total of thirty-two shrimp fractions were obtained from the ethyl acetate fraction and the water fraction obtained in Example 2, and 13 single fractions, which are considered to be effective for inflammation, were isolated.

The ethyl acetate fractions were used to isolate seven single fractions as follows.

First, to separate linarin, ethyl acetate fraction on silica gel was dissolved and separated using a column of dichloromethane and methanol, and the fraction was adjusted by TLC. Seven fractions were obtained with increasing methanol solvent. Thereafter, fractionation C. 7 was used to separate the linarin using a silica gel column using a mixed solvent of methanol and water.

Thereafter, the fraction C.5 was subjected to the same fractionation procedure as above using a column to obtain tricin, isorhamnetin-3-O-beta-D-glucopyranoside D-glucopyranoside) and apigenin-7-O-beta-D-glucopyranoside.

Further, the fraction C.4 was fractionated using a methanol / water mixture solvent using a column, and methyl 3, 5-di-OE-caffeoyl quinate (methyl 3 , 5-di-OE-caffeoyl-quinate), 36-A-CI-43D1_ new compound (36A-CI-43D1_new compound) and quercetin.

Then, the water fractions were used to separate the six single fractions as follows.

The water fractions were dissolved and separated by using methanol and water mixtures using a column. The fractions were adjusted by TLC and three fractions were obtained while increasing methanol solvent.

Thereafter, four fractions were obtained from the fraction D.2 using a mixed solvent of dichloromethane and methanol using a column. Then, the fractions were fractionated in the fraction D.2.3 using methanol and water and a mixed solvent of dichloromethane and methanol Quercetin-7-O- beta -D-glucopyranoside was isolated from the culture medium.

Six fractions were obtained from the fraction D.3 using a methanol and water mixture using a column. Then, the fractions D.3.5 were fractionated with methanol and water using a mixed solvent of dichloromethane and methanol to obtain 3-β- D-glucopyranosyl-3-epi-2-isocucurbitic acid, (+) - pinoresinol-4-O- β-D-glucopyranoside and β-D-glucopyranoside ((+) - pinoresinol-4-O-β-D-glucopyranoside and syringaresinol- ). C-58C_new compound (36A-CI-58C_new compound) and (-) - cis-chrysanthenol-β-D-catechol in the fraction D.3.6 using a mixed solvent of dichloromethane and methanol and methanol and water. Glucopyranoside ((-) - cis-chrysanthenol-β-D-glucopyranoside).

Experimental Example 1. Cell viability analysis

RAW 264.7 cells were used to analyze the cell viability of the hamster extract or fractions. Mouse macrophage cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin at 37, 5% CO ₂ . The cells were stabilized by subculturing two or more times.

1-1. Cell viability of hamster extract or fraction

In order to analyze the cell viability of the hamster extract and fractions obtained in Example 1 or 2, experiments were conducted using MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide) Respectively. After incubation for 24 hours at the above concentrations, the culture broth was changed. After 24 hours, the culture medium was changed, and then the extracts, ethyl acetate fraction and water fractions of 4, 20 and 100 Mu] g / ml and measured using MTT buffer after 24 hours. This experimental procedure was conducted with three sets of reference samples and samples. The cell viability was calculated by the following equation, and the results are shown in FIG.

[Mathematical Expression]

Cell survival rate (%) = (absorbance of sample treated group / absorbance of control group) x 100

Experimental Results As shown in Fig. 2, it was confirmed that the mugwort extract, ethyl acetate fraction and water fraction were not toxic at a high concentration of 100 μg / ml.

1-2. Cell viability of thirteen species

The cell viability of the 13 kinds of germinated single fractions obtained in Example 3 was measured at the concentrations of 0.4, 2, and 10 mu m, and the same procedure as described above was carried out. The results are shown in FIG.

As shown in FIG. 3, it was confirmed that the 13 single-section cut-off fractions obtained in Example 3 were not toxic at a high concentration of 10 μm.

Experimental Example 2: Inhibitory effect on nitrogen monoxide (NO) production of extract or fraction from hamster

2-1. Inhibitory effect of nitrogen extracts or fractions on nitrogen monoxide production

In order to measure the NO production inhibitory effect of the hamster extract and fractions obtained in Example 1 or 2, nitrite in the supernatant of RAW 264.7 cells cultured in Experimental Example 1 was measured to analyze NO production amount. Cells were inoculated into 24-well culture dishes at a concentration of 2 x ¹⁰ cells / well. Cells were pre-cultured, and the hamster extract and fractions obtained in the above Example 1 or 2 were pre-treated at a concentration of 4, 20 and 100 μg / ml, and after 1 hour, stimulated with LPS (lipopolysaccharide, 1 μg / Lt; / RTI > Then, the supernatant was mixed with the same volume as the Griess reagent, and the mixture was allowed to react at room temperature for 20 minutes. The concentration of nitrogen monoxide was measured at a wavelength of 520 nm, and the results are shown in FIG.

Experimental Results As shown in FIG. 4, it was confirmed that the hamster extract and fractions obtained in the above Example 1 or 2 inhibited the production of nitrogen monoxide in a concentration-dependent manner, in particular, the production of NO in the ethyl acetate fraction was remarkably inhibited, Statistically significant (* p <0.05, ** p <0.005).

2-2. Inhibitory effect of 13 kinds of fecal fractions on the production of nitrogen monoxide (NO)

In order to measure inhibition of NO production of the 13 kinds of germinated single fractions obtained in Example 3, experiments were conducted as described above, and the results are shown in Fig.

As shown in FIG. 5, among the thirteen kinds of demarcated single fractions obtained in Example 3, quesetin-7-O- beta -D-glucopyranoside and 3-beta -D- glucopyranosyl- - epo-2-isoxucurbitic acid, which was similar to that of celecoxib used as a conventional COX-2 inhibitor, and was statistically significant (* p < 0.05, ** p < 0.005).

EXPERIMENTAL EXAMPLE 3 Inhibition of Tumor Necrosis Factor (TNF-α) Expression in Extracts or Fractions of Cuttlefish

3-1. Inhibitory effect of TNF-α on extracts or fractions

In order to measure the TNF-α inhibitory effect of the hamster extract and fractions obtained in Example 1 or 2, RAW 264.7 cells cultured in Experimental Example 1 were inoculated in a 96-well culture dish at a concentration of 1 × 10 ⁵ cells / ml Respectively. Cells were pre-cultured, and the hamster extract and fractions obtained in Example 1 or 2 were pre-treated at a concentration of 4, 20 and 100 μg / ml, respectively. After 1 hour, the cells were pretreated with LPS (lipopolysaccharide, 1 μg / Lt; / RTI &gt; The concentration of TNF- [alpha] was then measured using an ELISA (Enzyme-Linked Immunosorbent Assay) kit (BD, Bioscience, USA). All experimental procedures were carried out at room temperature, and all reference samples and concentrations were repeated three times and the results are shown in FIG.

Experimental Results As shown in FIG. 6, it was confirmed that TNF-α expression was inhibited in the hamster extract and fractions obtained in Example 1 or 2 above. In particular, it was confirmed that TNF-α expression was significantly inhibited in the ethylacetate-ethyl acetate fraction, which was statistically significant (* p <0.05, ** p <0.005).

3-2. Inhibitory effect of TNF-α on 13 fractions

The inhibition of the expression of TNF- [alpha] by the 13 kinds of germ-line single fractions obtained in Example 3 was experimented as described above, and the results are shown in Fig.

As shown in FIG. 7, among the thirteen locust bean fractions obtained in Example 3, linarin, 3-β-D-glucopyranosyl-3-epi- The expression of TNF- [alpha] in the novel compounds was inhibited by pinoresinol-4-O- [beta] -D-glucopyranoside, cyclin resorcinol-O- [beta] -D- glucopyranoside and 36A-CI- (* P <0.05, ** p <0.005), which was similar to that of celecoxib used as a conventional COX-2 inhibitor.

Experimental Example 4. Western blot analysis of extracts and fractions

(KOX-2) and inducible nitric oxide synthase (iNOS) expression using western blot to confirm that the hamster extract and fractions obtained in Example 1 or 2 were inhibiting inflammation-related genes Inhibitory effect was confirmed.

4-1. Inhibitory effect of extracts on the COX-2 and iNOS expression

In order to measure COX-2 and iNOS inhibitory effects of the hamster extract obtained in Example 1, RAW 264.7 cells cultured in Experimental Example 1 were inoculated in a 6-well culture dish at a concentration of 2 x ¹⁰ cells / ml. After cell culture, the hamster extract obtained in Example 1 was treated at a concentration of 4, 20, 100 μg / ml and treated for 1 hour with LPS (1 μg / ml) stimulation for 24 hours. The cells were then removed and centrifuged, followed by lysis buffer to homogenize the cells, followed by centrifugation to obtain supernatants. This supernatant was loaded onto 12% SDS-polyacrylamide gel and blotted onto the membrane. The membranes were washed with 1 M Tris buffer (TBS, pH 7.2) supplemented with 0.1% Tween-20 and fixed in TBS-T supplemented with 5% skim milk for 2 hours. After washing with TBS-T, the anti-COX-2, iNOS-labeled antibody was diluted 1: 2500 in TBS-T supplemented with 5% skim milk. After overnight incubation at 4 ° C, the cells were washed with TBS-T, and the secondary antibody was diluted at a ratio of 1: 2000 and treated for 1 hour. Thereafter, the protein band was visualized using an enbanced chemiluminescence (ECL) system, and the results of COX-2 and iNOS Western blotting by hamster extract are shown in FIG.

As shown in FIG. 8, COX-2 and iNOS were strongly expressed in inducing group when inflammation was induced in RAW 264.7 cells by LPS, whereas in the group treated with extract of hamyang, which is an active ingredient of the present invention, COX-2 and iNOS expression were decreased.

4-2. Inhibitory effect of COX-2 and iNOS on the fraction of ethylacetate and water fraction

In order to measure the COX-2 and iNOS expression inhibitory effects of the gut steamed ethyl acetate fraction and the water fraction obtained in Example 2, experiments were conducted as described above, and the results are shown in FIGS.

Experimental Results As shown in FIG. 9, the expression of COX-2 and iNOS was significantly reduced at a concentration of 20 and 100 μg / ml of the Gamma ethyl acetate fraction of Example 2. In addition, as shown in Fig. 10, it was confirmed that the expression of COX-2 and iNOS was markedly reduced at a concentration of 100 占 퐂 / ml in the case of the red ginseng water fraction.

Experimental Example 5. Inflammation Inhibitory Effect on the Experimental Model of Inflammatory Bowel Disease

5-1. Build an experimental model

Seven weeks old male Balb / c male mice (20 ~ 25g) were purchased from Samtako (Osan, Korea) for freezing of solid feed and water for 1 week to establish an experimental model of inflammatory bowel disease. 5%), constant temperature (22 ± 2 ℃), and 12-hour period. After the adaptation period for one week, the groups were divided into 15 groups. The normal control group, acute colitis induced group (DSS), ganghwado ethanol extract treatment group (8, 40, 200mg / kg) ASA (2, 10, 50 mg / kg) as a positive control and 2, 10, 50 mg / kg as a control group acetyl salicylic acid) (100 mg / kg). The extracts, fractions and 5-ASA were dissolved in 0.9% saline, and then given orally once a day for 7 days at the same time as 5% DSS (dextran sulfate sodium). The normal control group and acute colitis induced group were 0.9% saline Orally. Acute colitis induced group, red feces fraction treated group and positive control group were freely consumed 5% DSS for 1 week, and normal control group was free to drink water. After 7 days, mice were sacrificed by cervical dislocation after ether anesthesia with ether, and the cecum from the cecum to the anus of the anus was excised. The length and appearance of the extracted colon were observed and scored according to the criteria in Table 1 below. After the colon contents were all removed, some of them were fixed in 10% formalin fixative for use as pathological tissue samples, and the remaining samples were stored frozen at -80 ° C for molecular biological analysis. Weight change, colonic length, disease activity index (DAI score and MPO activity) and western blot analysis were carried out as follows in the case of the colostrum extract in the colitis model, and the results are shown in FIGS. 11 to 14.

score Weight reduction (%) Concentration of feces Potential / severe bleeding 0 (-) usually usually One 1-5 2 5-10 Dilution Potential bleeding 3 11-15 4 > 15 diarrhea Severe bleeding

Disease activity index = (weight loss + concentration of stool + potential or severe bleeding) / 3

5-1. Weight change

Inflammatory Bowel Disease was induced in DSS and changes in body weight were checked once a day.

5-2. Colon length and DAI score

The length of the extracted colon was measured and the DAI scores according to the criteria in Table 1 are shown in the figure.

5-3. Inhibition of MPO (Myeloperoxidase) activity

23 mg of the colonic tissue of the experimental animals was homogenized in a 50 mM phosphate buffer containing 100 μl of 0.5% HTAB (hexadecyltrimethyl ammonium bromide), and then the supernatant was obtained using a centrifuge. Then, the supernatant, 50 mM phosphate buffer solution containing 0.5% HTAB, O-dianisidine, and 0.003% H ₂ O ₂ were added to a 96-well plate and the absorbance was measured three times at 450 nm for 5 minutes.

5-4. Western blot analysis

In order to confirm whether the extracts and fractions of Tamagi extract of the present invention inhibit the inflammation-related genes, we confirmed the expression of COX-2, iNOS and NF-kB p65 by Western blotting.

First, the colonic tissue was homogenized by adding a lysis buffer, and then the supernatant was obtained using a centrifuge. This was loaded on a 12% SDS-polyacrylamide gel and blotted onto the membrane. The membranes were washed with 1 M Tris buffer (TBS, pH 7.2) supplemented with 0.1% Tween-20 and fixed in TBS-T supplemented with 5% skim milk for 2 hours. After washing with TBS-T, the antibody labeled with anti-COX-2, iNOS and NF-kB p65 was diluted 1: 2500 in TBS-T supplemented with 5% skim milk. After overnight incubation at 4 ° C, the cells were washed with TBS-T, and the secondary antibody was diluted at a ratio of 1: 2000 and treated for 1 hour. The protein bands were then visualized using an enbanced chemiluminescence (ECL) system.

FIG. 11 shows the results of the experiment on the Ganoderma lucidum extract isolated from Example 1, showing that the weight loss rate was the lowest at 200 mg / kg of Ganoderma lucidum ethanol extract (FIG. 11A) (Fig. 11B). In particular, it was confirmed that the ethanol extract of 200 mu g / kg showed almost the same level as 5-ASA, which is a control drug group, in a statistically significant result (Fig. 11B). Also, as shown in FIG. 11C, it was confirmed that the DAI score was decreased as the concentration of ethanol extract of Ganoderma lucidum was increased. As shown in FIG. 11D, MPO activity was also decreased with increasing concentration. In addition, Western blot analysis of COX-2, iNOS and NF-kB p65 by ethanol extract of hamster showed that the expression of COX-2, iNOS and NF-kB p65 in 200 mg / kg of hamster ethanol extract Respectively.

FIG. 12 shows the results of the experiment on the Ganoderma lucidum extract isolated from Example 1, showing that the weight loss rate was the lowest at 200 mg / kg of Ganoderma lucidum extract (FIG. 12A) , Which was statistically significant (Figure 12B). Also, as shown in FIG. 12C, it was confirmed that the DAI score was decreased as the concentration of ethanol extract of Ganoderma lucidum was increased. As shown in FIG. 12D, the MPO activity was also decreased with increasing concentration.

FIG. 13 shows the results of the fractionation of the ganoderma ethyl acetate fraction isolated in Example 2, showing that the body weight reduction rate was the lowest at 50 mg / kg of the ganoderma ethyl acetate fraction (FIG. 13A) Acetate fraction increased, and the colonic length was slightly increased in comparison with 5-ASA, which is a control drug group, in 50 mg / kg of the ethmoguanium ethyl acetate fraction (FIG. 13B). As shown in FIG. 13C, the DAI score was decreased as the concentration of ethyl ethanoate fraction was increased, and the MPO activity was also decreased with increasing concentration as shown in FIG. 13D. In addition, the Western blotting results of COX-2, iNOS and NF-kB p65 by the ethidium ethyl acetate fraction showed that the expression of each was decreased as the concentration of the ethylacetate fraction was increased as shown in FIG. 13E, / Kg, the expression level of COX-2, iNOS and NF-kB p65 was significantly reduced, which was almost the same as or less than that of 5-ASA, the reference drug.

FIG. 14 shows that the weight loss rate was the lowest at 50 mg / kg of the Ganoderma lucidum fraction (FIG. 14A), which was similar to the 5-ASA as the reference drug group Respectively. In addition, the length of the extracted colon was increased as the concentration of the gut water fraction increased, and in particular, it was found to be almost the same as that of 5-ASA, which is the control drug group, at 50 mg / kg of the gut water fraction (Fig. 14B). As shown in FIG. 14C, the DAI score was decreased as the concentration of the water-soluble fraction was increased. Especially, 50 mg / kg of the water fraction of the immature lot was almost similar to 5-ASA as the reference drug. In addition, as shown in FIG. 14D, the MPO activity was decreased as the concentration of the gut water fraction increased, and the results of western blotting of COX-2, iNOS and NF-kB p65 also showed that the concentration of the water- , And the expression of COX-2, iNOS and NF-kB p65 was significantly reduced at 50 mg / kg, which was almost the same as or less than that of 5-ASA, the reference drug.

Formulation Example 1. Preparation of pharmaceutical preparations

Acid production

20 mg of hamsters extract or fractions, 100 mg of lactose and 10 mg of talt were mixed and filled in airtight bags to prepare powders.

Tablet manufacture

10 mg of hamsters extract or fraction, 100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate were mixed and tableted according to a conventional preparation method.

Capsule preparation

In accordance with the usual preparation method of capsule, 10 mg of hamsters extract or fraction, 3 mg of crystalline cellulose, 14.8 mg of lactose and 0.2 mg of magnesium stearate were mixed and filled in gelatin capsules to prepare capsules.

Injection manufacturing

According to the usual injection preparation method, 10 mg of hamchi extract or fraction per 1 ampoule (2 mL), 180 mg of mannitol, 2,974 mg of sterilized distilled water for injection and 26 mg of Na ₂ HPO ₄ .2H ₂ O were prepared.

Liquid preparation

In accordance with the usual method for preparing a liquid preparation, 20 mg of gypsum extract or fraction, 10 g of isomerized sugar and 5 g of mannitol were added to purified water and dissolved, and the lemon flavor was added in an appropriate amount. Then, purified water was further added thereto, adjusted to a total volume of 100 mL, filled in a brown bottle, and sterilized to prepare a liquid preparation.

Formulation Example 2. Preparation of food preparation

Health food manufacturing

100 mg vitamin C acetate, 70 mg vitamin A acetate, 0.13 mg vitamin B, 0.15 mg vitamin B2, 0.5 mg vitamin B6, 0.2 g vitamin B12, 10 mg vitamin C, 10 g biotin, nicotinic acid Amide 1.7 mg, folate 50 g, calcium pantothenate 0.5 mg, an appropriate amount of mineral mixture, ferrous sulfate 1.75 mg, zinc oxide 0.82 mg, magnesium carbonate 25.3 mg, potassium phosphate monobasic 15 mg, dibasic calcium phosphate 55 mg, potassium citrate 90 Mg of calcium carbonate, 100 mg of calcium carbonate, and 24.8 mg of magnesium chloride were mixed and granules were prepared and a health food was prepared according to a conventional method. At this time, although the composition ratio of the vitamin and mineral mixture is relatively mixed with the ingredient suitable for health food, it may be arbitrarily modified.

Health drink manufacturing

According to a conventional method for producing healthy beverages, 100 mg of green tea extract or fraction, 15 g of vitamin C, 100 g of vitamin E (powder), 19.75 g of iron lactate, 3.5 g of zinc oxide, 3.5 g of nicotinic acid amide, 0.2 g of vitamin A, , 0.3 g of vitamin B2 and a predetermined amount of water were mixed and heated at 85 DEG C for about 1 hour with stirring. The resulting solution was filtered to obtain a sterilized 2 L container, sealed sterilized and refrigerated to prepare a health drink. At this time, although the composition ratio of the ingredients suitable for the beverage is comparatively mixed, the mixture ratio may be arbitrarily varied according to the demand, the demanded country, the intended use, and the regional or national preference.

Although the present invention has been described in terms of the preferred embodiments mentioned above, it is possible to make various modifications and variations without departing from the spirit and scope of the invention. It is also to be understood that the appended claims are intended to cover such modifications and changes as fall within the scope of the invention.

Claims (14)

A pharmaceutical composition for preventing or treating an inflammatory disease comprising an ethyl acetate fraction obtained by sequentially fractionating a 70% (v / v) ethanol extract of Degussaukrug with dichloromethane, ethyl acetate and water. delete delete delete delete delete The method according to claim 1,
Wherein the ethyl acetate fraction is contained in an amount of 0.01 to 95% by weight based on the total weight of the composition. The method according to claim 1,
Wherein the ethyl acetate fraction reduces the concentration of cyclooxygenase (COX-2), tumor necrosis factor (TNF-a) or myeloperoxidase (MPO) &Lt; / RTI &gt; The method according to claim 1,
Wherein the ethyl acetate fraction reduces the expression level of nitrogen monoxide (NO) or inducible nitric oxide synthase (iNOS). The method according to claim 1,
Wherein said ethyl acetate fraction inhibits the nuclear factor kappa B (NF-kB) activity. The method according to claim 1,
Wherein said ethyl acetate fraction inhibits inflammation in macrophages. &Lt; RTI ID = 0.0 &gt; 21. &lt; / RTI &gt; The method according to claim 1,
The inflammatory disease may be selected from the group consisting of gastritis, gastric ulcer, bronchitis, asthma, edema, hepatitis, nephritis, atherosclerosis, inflammatory bowel disease (IBD), colitis, rheumatoid arthritis, osteoarthritis, Or a pharmaceutically acceptable salt thereof. A food composition for the improvement of inflammation diseases, comprising an ethyl acetate fraction obtained by sequentially fractionating 70% (v / v) ethanol extract of Degussa gut using dichloromethane, ethyl acetate and water. 14. The method of claim 13,
Wherein the ethyl acetate fraction is contained in an amount of 0.01 to 95% by weight based on the total weight of the composition.

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