KR20120077075A - Composition for improving allergy disease using the effective compound which cay be isolated from an extract of wheat bran - Google Patents

Abstract

PURPOSE: A composition containing a compound isolated from wheat rice bran extract is provided to suppress degranulation and cytokine generation. CONSTITUTION: A composition for treating allergic diseases contains 5-n-nonadecylresorcinol of chemical formula 1 or 5-n-heicosylresorcinol of chemical formula 2 as an active ingredient. The composition is a pharmaceutical, food, or cosmetic composition. A composition for treating inflammatory diseases contains the compound of chemical formula 1 or 2 as an active ingredient. The pharmaceutical composition is manufactured in the form of an oral formulation, parenteral formulation, and topical formulation.

Description

Composition for Improving Allergy Disease Using the Effective Compound Which Cay Be Isolated from an Extract of Wheat Bran}

The present invention relates to an allergic disease improver composition using an active substance that can be separated from wheat bran extract.

Allergy is a reaction caused by immunological mechanisms caused by allergen, which is a foreign substance, which generally causes harmful damage to the body.

Mast cells are important cells that mediate allergic reactions. They are widely distributed throughout the organs of the body, such as the skin, respiratory organs, mucous membranes of the gastrointestinal tract, around lymphatic vessels, around blood vessels, and in the brain, and have rich granules in the cytoplasm.

The allergic reaction is caused by the action of IgE on mast cells against house dust mites, pollen, animal dander, mold, egg albumin, milk proteins, and peanuts (Wills-Karp M., et al., Science, 282, pp 2258-61, 1998; Grunig G., et al., Science, 282, pp 2261-2263, 1998).

When allergens invade a living body, allergens are first recognized by antigen presenting cells, which differentiate Th0 into Th2 cells by presenting allergens in the presence of IL-4. These differentiated Th2 cells secrete cytokines such as IL-4, IL-5, IL-13, IL-4, IL-5 and the like act on B cells to induce the production of IgE (Kato, Y. et al., J. Immuno. 1999, 162, 7470-7479; Toru, H. et al., J. Allergy. Clin. Immunol. 1998, 102, 491-502).

IgE binds to the high-affinity IgE receptor, FcεRI, in mast cells.Allergens bind to IgE, which is bound to FcεRI in mast cells, causing cross-linking of these receptors, which acts as a signal to trigger a series of signaling reactions. (Marshall, JS Nat. Rev. Immunol. 2004, 4, 787-799).

When the mast cell is activated through this signaling reaction, histamine, proteoglycan, and TNF-α, which are chemical mediators present in the cell granules, are secreted, and leukotrienes are released through the arachidonic acid metabolism liberated from cell membrane phospholipids. (leukotriens) and prostaglandins, such as inflammatory mediators (Nadler, MJ et al., Adv. Immunol. 2004, 76, 325-355). It is known to mediate a series of allergic inflammatory responses, including stimulation of nerve endings, increased mucus secretion, and contraction of smooth muscle (Meltzer, EO et al., Ann Allergy Asthma Immunnol. 2000, 84, 176-187).

The fact that antihistamines, leukotriene antagonists, anti-IgE antibodies, substances that inhibit the synthesis and secretion of IgE (such as diacylbenzimidazole homologues disclosed in US Pat. No. 6,369,091, etc.) may improve allergic symptoms. Support the mechanism.

The present invention is completed by confirming that the active substance that can be separated into wheat bran extract inhibits the activation of mast cells.

An object of the present invention is to provide a composition for improving allergic diseases.

Other and specific objects of the present invention will be presented below.

The inventors have identified 5-n-nonadecyl resorcinol and 5-n-henecosilosesi isolated from Wheat Bran extract, as confirmed in the following Examples and Experimental Examples. Treatment of mast cell line RBL-2H3 (rat basophilic leukemia) activated with IgE and antigen (DNP-BSA) inhibited the production of beta-hexosaminidase by 5-n-heneicosylresorcinol Could confirm. The mast cell line RBL-2H3 has a receptor of IgE antibody (FcεRI) on its surface, and when activated with IgE and antigen, it is a cell known to secrete allergic mediators such as histamine by degranulation. Sosaminidase is a substance known as an indicator of degranulation (J. Korean Soc. Appl. Biol. Chem. 48 (4), 315-321 (2005)). The inventors also note that allergic and inflammatory reactions, such as 5-n-nonadesilesocinol and 5-n-henecoselezosinol TNF-α, IL-4, IL-6, IL-10, isolated from wheat bran extract It was confirmed that the production of cytokines involved in the inhibition of, and further inhibits the production of p-AKT. p-AKT is known to be involved in mast cell proliferation and cytokine production via phosphoinositol 3-kinase (PI3K) (Huang, F. et al., Int. Immunopharmacol. 2008, 8, 502-507) .

The present invention has been completed on the basis of the experimental results described above, the allergic disease improver composition of the present invention is 5-n- nonadesilesocinol or 5-n-henecosyl which can be isolated from wheat bran extract It is characterized by including the josinol as an active ingredient.

The 5-n- nonadecyl having silane and 5-n- play Jos H. cat silane is described by Joshi play (J Bacteriol 1996 July; 178 ( 14):.. 4027-4030; J. Agric Food Chem . 2003, 51 , 6683-6688; Chem. Pharm . Bull . 1989, 37 , 2431-2434; Biosci . Biotech . Biochem . 1997, 61 , 480-486) and the like are known, its IUPAC name and its chemical formula can be found in the following [Formula 1] and [Formula 2], respectively.

In the present specification, the "wheat bran" means a by-product obtained in the process of milling wheat. These by-products are usually composed of the rind (seed shell) and the embryo, except for the endosperm, which is flour, or when the embryo is separated with the endosperm during milling of wheat. Wheat bran can contain small amounts of embryos and embryos. The bran powder obtained by crushing wheat bran may be further subdivided into bran, powder, shank, isocracker, etc. according to the particle size, and in the present specification, "bran bran" is used to remove the bran, powder, shank, isocrape, etc. derived from such bran powder. It is meant to include.

In the present specification, the "extract" means lower alcohols having 1 to 4 carbon atoms such as water, methanol, ethanol, butanol, ethylene, acetone, n-hexane, ether, chloroform, ethyl acetate, butyl acetate, dichloromethane, N, Fractional crude extract and its extract obtained using N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, or a mixed solvent thereof are fractionated into one or more of the solvents listed above. Refers to the fraction obtained by. The extraction method may be applied in any manner, such as cooling, reflux, heating, ultrasonic radiation in consideration of the degree of extraction, the degree of preservation of the active ingredient. In the case of fractionated extracts, the crude extract is suspended in a specific solvent and mixed with a solvent having a different polarity. The crude extract is adsorbed onto a column filled with silica gel and the like, followed by a hydrophobic solvent, a hydrophilic solvent, or a mixture thereof. It is meant to include a fraction obtained by using a solvent as a mobile phase. In addition, the meaning of the extract includes a concentrated liquid extract or solid extract in which the extraction solvent is removed in a manner such as freeze drying, vacuum drying, hot air drying, spray drying, and the like. Preferably, the "extract" means an extract obtained by using ethanol as an extraction solvent or a fraction of an n-hexane layer obtained by solvent fractionation of the ethanol extract with water and n-hexane.

In addition, in the present specification, the meaning of the "active ingredient" means a component that can exhibit activity alone or in combination with a carrier that is not active alone.

In the present specification, the "allergic disease" is caused by activation of mast cells such as degranulation of mast cells. Refers to clinical symptoms caused by allergic reactions, which may include contact dermatitis, atopic dermatitis, eczema, pruritus, hay fever, asthma, bronchitis, urticaria, vasculitis, rhinitis, gastroenteritis, diarrhea, Interstitial pneumonia, arthritis, ophthalmitis, conjunctivitis, neuritis, otitis media, granulomatosis, encephalomyelitis, cystitis, laryngitis, purpura, food allergy, insect allergy, drug allergy, metal allergy, anaphylactic shock (Bierman CW, et al. (Eds.) Allergy, asthma, and immunology from infancy to adulthood. Page xvii, Saunders, Philadelphia, 1996).

In addition, in the present specification, the "improvement" means the prevention, treatment, inhibition of onset, or delay of pathological symptoms.

On the other hand, the composition of the present invention is any amount as long as it can exhibit the improvement activity of the allergic disease intended to treat the compound of [Formula 1] or [Formula 2], the active ingredient according to the use, formulation, formulation purposes, etc. Effective amount), a conventional effective amount will be determined within the range of 0.001% to 15% by weight, based on the total weight of the composition. An "effective amount" as used herein refers to an amount of an active ingredient in a mammal, preferably a human subject to which it is applied, that can induce improvement, treatment, or suppression / delay of the development of such pathological symptoms. Such effective amounts can be determined experimentally within the range of ordinary skill in the art.

In another aspect, the present invention relates to an inflammatory disease improving composition comprising the compound of [Formula 1] or [Formula 2] as an active ingredient.

IgE and its antigen-activated mast cells not only secrete histamine, which causes allergic reactions by degranulation, but also prostaglandins, leukotrienes, TNF-α, IL-1β, IL-4, IL-5, IL-6, Cytokines such as IL-13 are synthesized and secreted. Prostagladins and leukotrienes are inflammatory mediators produced from arachidonic acid by COX or lipoxygenase, and TNF-α, IL-1β, IL-6 and the like are representative inflammatory cytokines (Thomas. R. , Rev. Physiol.Biochem.Pharmacol., 100, 1984). The prostaglandins and the like promote the migration of leukocytes to the site of inflammation, and the inflammatory cytokines induce the expression of iNOS producing NO which causes cytotoxicity and various inflammatory reactions and the expression of COX-2 involved in inflammation and pain. . IL-3, IL-5 and IL-13 are also known to cause infiltration and activation of eosinophils that mediate chronic allergic inflammation (Erb KJ., Immunol. Today, 20, pp 317-322, 1999; Rothenberg ME., Eosinophilia., N. Engl. Med., 338, pp 1592-1600, 1998).

Experimental Example below shows that the compound of [Formula 1] or [Formula 2] not only inhibits the production of inflammatory cytokines of activated mast cells but also inhibits expression of COX-2. And inhibits the production of NF-κB, an inflammatory cytokine transcription factor, and the inhibition of the production of mitogen-activated protein kinase (MAPK) (ERK, JNK and p-38), which are involved in the activation of NF-κB. It also shows that it inhibits the activity of lipoxygenase.

These experimental results can be said to show that the compound of [Formula 1] or [Formula 2] can be usefully used as an inflammatory disease improver.

In the present specification, the "inflammatory disease" may be defined as any condition specified by a local or systemic biological defense response against external physical and chemical stimuli or infection of an external infectious agent such as bacteria, fungi, viruses, and various allergens. This response activates various inflammatory mediators and enzymes associated with immune cells (eg iNOS, COX-2, etc.), secretion of inflammatory mediators (eg NO, TNF-α, IL-6, IL-1β, PGE ₂₎ . ), Accompanied by a series of complex physiological reactions such as fluid infiltration, cell migration, tissue destruction, etc., and are manifested externally by symptoms such as erythema, pain, edema, fever, deterioration or loss of certain functions of the body. The inflammatory disease may be acute, chronic, ulcerative, allergic or necrotic, so as long as any disease is included in the definition of such inflammatory disease it is acute, chronic, ulcerative, allergic or Irrespective of necrosis Specifically, the inflammatory diseases include asthma, allergic and non-allergic rhinitis, chronic and acute rhinitis, chronic and acute gastritis or enteritis, ulcerative gastritis, acute and chronic nephritis, acute and chronic hepatitis, chronic obstructive pulmonary disease, pulmonary fibrosis Irritable bowel syndrome, inflammatory pain, migraines, headache, back pain, fibromyalgia, fascia disease, viral infections (eg, type C infections), bacterial infections, fungal infections, burns, surgical or dental wounds, Prostaglandin E excess syndrome, atherosclerosis, gout, arthritis, rheumatoid arthritis, ankylosing spondylitis, Hodgkin's disease, pancreatitis, conjunctivitis, irisitis, scleritis, uveitis, dermatitis (including atopic dermatitis), eczema, multiple sclerosis, etc. Will be included.

In the improver composition of the inflammatory disease of the present invention, as described in connection with the improver composition of the allergic disease of the present invention, the meanings of the improvement, the effective amount and the like are effective as they are.

The allergic disease improver composition of the present invention and the inflammatory disease improver composition (hereinafter “composition of the present invention”) may be used as pharmaceutical compositions in specific embodiments.

The pharmaceutical composition of the present invention includes oral formulations (tablets, suspensions, granules, emulsions, capsules, syrups, etc.), parenteral formulations (sterile injectable aqueous or Oily suspensions), topical formulations (solutions, creams, ointments, gels, lotions, patches) and the like.

As used herein, "pharmaceutically acceptable" means that the subject of application (prescription) does not have more toxicity (adequately low toxicity) to which the subject of application (prescription) is adaptable without inhibiting the activity of the active ingredient.

Examples of pharmaceutically acceptable carriers include lactose, glucose, sucrose, starch (such as corn starch, potato starch, etc.), cellulose, derivatives thereof (such as sodium carboxymethyl cellulose, ethylcellulose, etc.) malt, gelatin, talc, solids Lubricants (e.g. stearic acid, magnesium stearate, etc.), calcium sulfate, vegetable oils (e.g. peanut oil, cottonseed oil, sesame oil, olive oil, etc.), polyols (e.g. propylene glycol, glycerin, etc.), alginic acid, emulsifiers (e.g. TWEENS), wetting agents (e.g. Sodium lauryl sulfate), colorants, flavoring agents, tableting agents, stabilizers, antioxidants, preservatives, water, saline, phosphate buffer solutions and the like. The carrier may be selected from one or more of suitable pharmaceutical formulations according to the formulation of the pharmaceutical composition of the present invention.

Excipients may be selected and used according to the formulation of the pharmaceutical composition of the present invention, for example, when the pharmaceutical composition of the present invention is prepared with an aqueous suspending agent, suitable excipients are sodium carboxymethyl cellulose, methyl cellulose, hydropropylmethylcellulose And suspending agents and dispersing agents such as sodium alginate and polyvinylpyrrolidone. Suitable excipients when prepared from injection solutions include Ringer's solution, isotonic sodium chloride, and the like.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and in some cases, can be administered topically.

The daily dose of the pharmaceutical composition of the present invention is usually 0.001 to 150 mg / kg body weight, and may be administered once or several times. However, since the dosage of the pharmaceutical composition of the present invention is determined in view of various related factors such as the route of administration, the age, sex, weight of the patient, the severity of the patient and the like, the dosage may limit the scope of the present invention in any aspect. It should not be understood as.

The composition of this invention can be grasped | ascertained as a food composition in another specific aspect.

The food composition of the present invention may be prepared as a dietary supplement, a special nutritional supplement, a functional beverage, and the like.

The food composition of the present invention may include sweeteners, flavoring agents, bioactive ingredients, minerals, etc. in addition to the active ingredients.

Sweeteners may be used in amounts that give the food a suitable sweet taste, and may be natural or synthetic. Preferably, natural sweeteners are used. Examples of natural sweeteners include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose and maltose.

Flavoring agents can be used to enhance the taste or aroma, both natural and synthetic. Preferably, a natural one is used. When using natural ones, the purpose of nutritional fortification can be performed in addition to the flavor. The natural flavor may be obtained from apples, lemons, citrus fruits, grapes, strawberries, peaches, and the like, or may be obtained from green tea leaves, round leaves, jujube leaves, cinnamon, chrysanthemum leaves, jasmine and the like. Also, those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, banks and the like can be used. The natural flavoring agent may be a liquid concentrate or a solid form of extract. In some cases, synthetic flavoring agents may be used, and synthetic flavoring agents may include esters, alcohols, aldehydes, terpenes, and the like.

As the physiologically active substance, catechins such as catechin, epicatechin, gallocatechin, epigallocatechin, vitamins such as retinol, ascorbic acid, tocopherol, calciferol, thiamine, riboflavin, and the like can be used.

As minerals, calcium, magnesium, chromium, cobalt, copper, fluoride, germanium, iodine, iron, lithium, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, silicon, sodium, sulfur, vanadium, zinc and the like can be used.

In addition, the food composition of the present invention may contain a preservative, an emulsifier, an acidulant, a thickener, and the like, in addition to the sweetener.

Such preservatives, emulsifiers and the like are preferably added and used in very small amounts as long as the use to which they are added can be achieved. By trace amount is meant numerically expressed in the range of 0.0005% to about 0.5% by weight based on the total weight of the food composition.

Examples of preservatives that can be used include sodium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), and the like.

Emulsifiers that can be used include acacia gum, carboxymethylcellulose, xanthan gum, pectin and the like.

Examples of acidulants that may be used include lead acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, and the like. Such acidulant may be added so that the food composition is at an appropriate acidity for the purpose of inhibiting the growth of microorganisms in addition to the purpose of enhancing taste.

Thickeners that can be used include suspending implements, sedimenters, gel formers, swelling agents and the like.

The composition of this invention can be grasped | ascertained as a cosmetic composition in a specific aspect.

When the composition of the present invention is conceived as a cosmetic composition, the cosmetic composition can be prepared in various forms, for example, emulsions, lotions, creams (oil-in-water, water-in-oil, multiphase), solutions, suspensions (anhydrous and Aqueous), anhydrous products (oil and glycol based), gels, masks, packs, powders and the like.

The composition of the present invention may include an acceptable carrier in a cosmetic preparation in addition to including the active ingredient.

Herein, "acceptable carrier in cosmetic preparation" means a compound or composition already known and used that can be included in cosmetic preparations or a compound or composition which will be developed in the future and does not have more toxicity than the body can adapt to when contacted with the skin.

The carrier may be included in the composition of the present invention from about 1% to about 99.99% by weight, preferably from about 50% to about 99% by weight of the composition.

However, it is understood that the ratios limit the scope of the present invention in any aspect because the ratios vary depending on the formulation of the cosmetics as described above and their specific application site (face or hand) or their preferred application amount. It should not be.

Meanwhile, examples of the carrier include alcohols, oils, surfactants, fatty acids, silicone oils, wetting agents, moisturizers, viscosity modifiers, emulsions, stabilizers, sunscreen agents, colorants, fragrances, and the like.

Alcohols, oils, surfactants, fatty acids, silicone oils, wetting agents, humectants, viscosity modifiers, emulsions, stabilizers, sunscreens, colorants, compounds / compositions that can be used as fragrances, etc., which can be used as the carrier are already known in the art. As a result, those skilled in the art can select and use appropriate materials / compositions.

As described above, according to the present invention, it is possible to provide a composition for improving allergic diseases. In addition, according to the present invention can provide a composition for improving inflammatory diseases. The allergic disease improver composition of the present invention and the inflammatory disease improver composition may be commercialized as drugs, functional foods, cosmetics and the like.

1 is a schematic diagram of a process for separating active substances from wheat bran extract.
2 is an ESI-MS spectrum of the compound represented by [Formula 1].
3 is a ¹ H-NMR spectrum of the compound of [Formula 1].
4 is ¹³ C-NMR spectrum of the compound of [Formula 1].
5 is an ESI-MS spectrum of the compound of [Formula 2].
6 is a ¹ H-NMR spectrum of the compound of [Formula 2].
7 is ¹³ C-NMR spectrum of the compound of [Formula 2].
8 is a result showing the degranulation inhibitory activity of the active material isolated from wheat bran extract.
9 is a result showing the cytokine production inhibitory activity of the active substance isolated from wheat bran extract.
FIG. 10 shows the results of suppressing the expression of NF-κB, a cytokine transcription factor of an active substance isolated from wheat bran extract.
11 is a result showing the MAPK expression inhibitory activity and p-AKT expression inhibitory activity of the active material isolated from wheat bran extract.

Hereinafter, the present invention will be described with reference to Examples and Experimental Examples. However, the scope of the present invention is not limited to these examples and experimental examples.

< Example > Push  From extract Anti-allergy  Isolation and Identification of Active Substances Having Activity

< Example  1> Push  Separation of Active Substances from Extracts

Wheat ( Triticum Ethanol was added to the wheat bran, a byproduct of aestivum L.), and extracted by cold sedimentation three times for 24 hours. After the filtrate was filtered, the filtrate was used to obtain an ethanol extract using a vacuum concentrator. The extract was suspended in distilled water, and then fractionated with n-hexane, followed by solvent fractionation with CH ₂ Cl ₂ . These by fractions evaporated under reduced pressure and hexane fractions showing the anti-allergic activity in obtained an extract with hexane to silica gel _{chromatography: CH 2 Cl 2 (5:} 1, 1: 1, 0: 1), CH 2 Cl 2: methanol (80: 1, 50: 1, 25: 1, 10: 1, 5: 1, 2: 1) and divided into 10 fractions (G36H-3-1 ~ 10), and a small fraction G36H-3-10 For 90% methanol and hexane was partitioned off to obtain a fraction 90% methanol extract G36H-4-3. Silicagel chromatography on fraction G36H-4-3 was carried out on mobile phase hexanes: acetone (1: 0, 90: 1, 70: 1, 50: 1, 30: 1, 10: 1, 5: 1, 3: 1, 2 : 1) and divided into 11 fractions (G36H-6-1 ~ 11) and prep. From small fraction G36H-6-3. Compounds 1 (G36H-9-4) and 2 (G36H-9-5) were separated using HPLC (YMC-ODS, 95-100% MeCN, 20 ml / min).

A schematic diagram of the separation process of the active material is shown in FIG. 1.

< Example  2> Identification of active substances

Example 2-1 Identification of Compound 1

The separated compound 1 was subjected to physicochemical and spectroscopic analysis.

White amorphous powder; Negative mode (ESI-MS) m / z 375 [M-H] ^- , 751 [2M-H] ^- ; ¹ H-NMR (CDCl ₃ + CD ₃ OD, 500 MHz) δ 6.20 (2H, d, J = 2.5 Hz, H-4, 6), 6.14 (1H, t, J = 2.5 Hz, H-3), 2.46 (2H, t, J = 7.5 Hz, H-1 '), 1.56 (2H, m, H-2'), 1.22-1.30 (32H, m, H-3 ', 4', 5 ', 6' , 7 ', 8', 9 ', 10', 11 ', 12', 13 ', 14', 15 ', 16', 17 ', 18'), 0.88 (3H, t, J = 7.5 Hz, H -19 '); ¹³ C-NMR (CDCl ₃ + CD ₃ OD, 125 MHz) δ 157.5 (C-1, 3), 145.7 (C-5), 107.2 (C-4, 6), 99.9 (C-2), 36.0 ( C-1 '), 31.9 (C-17'), 31.2 (C-2 '), 29.4-29.7 (C-3', 4 ', 5', 6 ', 7', 8 ', 9', 10 ', 11', 12 ', 13', 14 ', 15', 16 '), 22.7 (C-18'), 14.0 (C-19 ')

Compound 1 as an amorphous powder in the form of a white ESI-MS [m / z 375 (M - H) -, 751 (2M - H) -] molecular weight (Fig. 2) were identified as 376 amu, molecular formula C ₂₅ H ₄₄ It was found that the O ₂ , the degree of unsaturation was 4. ^{In the 1} H-NMR spectrum (FIG. 3), three aromatic protons [δ _H 6.20 (2H, d, J = 2.5 Hz, H-4, 6), 6.14 (1H, t, J = 2.5 Hz, H-3) ], Alkyl group protons [δ _H 2.46 (2H, t, J = 7.5 Hz, H-1 '), 1.56 (2H, m, H-2'), 1.22-1.30 (32H, m), 0.88 (3H, t, J = 7.5 Hz, H-19 ')]. ^{In 13} C-NMR (FIG. 4), a total of 25 carbon signals including a signal derived from a benzene ring and an alkyl group were identified. From the above spectroscopic data and the degree of unsaturation of Compound 1 was estimated to be reported isolated from wheat 5-alkylresorcinol-based compound, a literature (J. Agric. Food Chem . 2003, 51 , 6683-6688; Chem . Pharm . Bull . 1989, 37 , 2431-2434; Biosci. Biotech . Biochem . 1997, 61 , 480-486) and the structure was determined by the 5-n-nonadecylresorcinol of [Formula 1].

Example 2-2 Identification of Compound 2

The separated compound 2 was subjected to physicochemical and spectroscopic analysis.

White amorphous powder; Positive mode (ESI-MS) m / z 405 [M + H] ⁺ ; (negative mode) m / z 403 [M−H] ⁻ ; ¹ H-NMR (CDCl ₃ + CD ₃ OD, 500 MHz) δ 6.20 (2H, d, J = 2.5 Hz, H-4, 6), 6.14 (1H, t, J = 2.5 Hz, H-3), 2.46 (2H, t, J = 7.5 Hz, H-1 '), 1.56 (2H, m, H-2'), 1.24-1.30 (36H, m, H-3 ', 4', 5 ', 6' , 7 ', 8', 9 ', 10', 11 ', 12', 13 ', 14', 15 ', 16', 17 ', 18', 19 ', 20'), 0.88 (3H, t, J = 7.5 Hz, H-21 '); ¹³ C-NMR (CDCl ₃ + CD ₃ OD, 125 MHz) δ 157.5 (C-1, 3), 145.6 (C-5), 107.1 (C-4, 6), 99.8 (C-2), 36.0 ( C-1 '), 31.9 (C-19'), 31.2 (C-2 '), 29.3-29.7 (C-3', 4 ', 5', 6 ', 7', 8 ', 9', 10 ', 11', 12 ', 13', 14 ', 15', 16 ', 17', 18 '), 22.7 (C-20'), 14.0 (C-21 ')

Compound 2 as an amorphous powder in the form of a white ESI-MS [m / z 405 (M + H) +, 403 (M - H) -] molecular weight (Fig. 5) was identified as 404 amu, molecular formula C ₂₇ H ₄₈ It was found that the O ₂ , the degree of unsaturation was 4. ^{In the 1} H-NMR spectrum (FIG. 6), three aromatic protons [δ _H 6.20 (2H, d, J = 2.5 Hz, H-4, 6), 6.14 (1H, t, J = 2.5 Hz, H-3) ], Alkyl group protons [δ _H 2.46 (2H, t, J = 7.5 Hz, H-1 '), 1.56 (2H, m, H-2'), 1.24-1.30 (36H, m), 0.88 (3H, t, J = 7.5 Hz, H-21 ')]. ^{In 13} C-NMR (FIG. 7), a total of 27 carbon signals including a signal derived from a benzene ring and an alkyl group were identified. Further, from the above spectroscopic data and the degree of unsaturation of compound 2 was estimated by separating the reported 5-alkylresorcinol-based compound from the mill, the literature (J. Agric. Food Chem . 2003, 51 , 6683-6688; Chem . Pharm . Bull . 1989, 37 , 2431-2434; Biosci . Biotech . Biochem . 1997, 61 , 480-486) and the structure was determined by the 5-n-heneicosylresorcinol of [Formula 2].

< Experimental Example > Push  Of the active substance isolated from the extract Anti-allergy  And anti-inflammatory activity experiment

< Experimental Example  1> Push  Of the active substance isolated from the extract Mast cell line Degranulation  Inhibitory Activity Experiment-β- Hexosaminidase assay

RBL-2H3 cell line, a rat basophlic leukemia cell line, was dispensed in ₂ × 10 ⁵ / well in a 24 well plate at MEM medium containing 15% FBS, 100 units / ml penicillin and streptomycin, and 37, 5% CO _2. Incubated for 24 hours.

After removing the supernatant, add MEM medium containing 25 ng / ml anti-DNP IgE, incubate for 4 hours, wash twice with 500 μl PIPES buffer, and wash PIPES buffer containing 4 mM MgCl ₂ , 5.6 mM glucose, and 0.1% BSA. Μl was incubated for 10 minutes. After pre-culture, the samples were treated for 20 minutes by concentration, and then reacted for 20 minutes at 50ng / ml DNP-BSA. After the reaction was terminated by 10 minutes on ice, the supernatant was added to a 96-well plate with 25 µl each, and 25 µl of 1 mM P-nitrophenyl-acetyl-β-D-glucosaminid was reacted at 37 ° C for 1 hour. . Stop solution (0.1M NaHCO ₃ , 0.1M Na ₂ CO ₃ ) was added to terminate the reaction and the absorbance was measured by an ELISA reader in the wavelength range of 405nm. Ketotifen was used as a positive control.

The results are shown in [FIG. 8]. Referring to [FIG. 8], it can be seen that all of the separated effective substances inhibit degranulation in a concentration-dependent manner. Wherein the EC ₅₀ of Compound 1 was found to be 243.71 uM and the EC ₅₀ of Compound 2 was found to be 174.39 uM.

< Experimental Example  2> Inhibitory Activity of Cytokine Production- RT-PCR

BL-2H3 cells were incubated in 6 well plates with 1X10 ⁶ / well, and then IgE was added and reacted for 4 hours. After that, the sample was treated for each concentration, and DNP-BSA was added and reacted at 37 ° C. for 20 minutes. After washing twice with PBS, Trizol (Invitrogen) was added to lyse the cells, chloroform (Sigma) was added and centrifuged at 13,000 rpm for 15 minutes to obtain a supernatant. After mixing the supernatant and isopropanol and reacting to precipitate RNA, the RNA was isolated by washing once with ethanol.

After quantification using the extracted RNA, cDNA was synthesized by reverse transcriptase chain polymerization using Superscript III (Invitrogen), a reverse transcriptase enzyme. PCR conditions were amplified DNA by 35 cycles of 94 ℃ 45 seconds, 55 ℃ 45 seconds, 72 ℃ 1 minutes 30 seconds. Gene expression inhibition was compared on agarose gel. The primers used are shown in [Table 1] below, and the control gene was GAPDH. Ketotifen was used as a positive control.

primer rCOX-2 F: 5 'TGT ATG CTA CCA TCT GGC TTC GG 3' (SEQ ID NO: 1) R: 5 'GTT TGG AAC AGT CGC TCG TCA TC 3' (SEQ ID NO: 2) rTNF-α F: 5 'CAC CAC GCT CTT CTG TCT ACT GAA C 3' (SEQ ID NO: 3) R: 5 'CCG GAC TCC GTG ATG TCT AAG TAC T 3' (SEQ ID NO: 4) rIL-4 F: 5 'ACC TTG CTT CAC CCT GTT C 3' (SEQ ID NO: 5) R: 5 'TTG TGA GCG TGG ACT CAT TC 3' (SEQ ID NO: 6) rIL-6 F: 5 'GCC CTT CAG GAA CAG CTA TGA 3' (SEQ ID NO: 7) R: 5 'TGT CAA CAA CAT CAG TCC CAA GA 3' (SEQ ID NO: 8) rIL-10 F: 5 'TAG AAG TGA TGC CCC AGG 3' (SEQ ID NO: 9) R: 5 'TCA TCC TCC ACC TGC TCC ACT GC 3' (SEQ ID NO: 10) GAPDH F: 5 'GAG TCA ACG GAT TTG GTC GT 3' (SEQ ID NO: 11) R: 5 'GAC AAG CTT CCC GTT CTC AG 3' (SEQ ID NO: 12)

The results are shown in [FIG. 9]. Referring to FIG. 9, it can be seen that all of the separated effective substances inhibit the production of cytokines in a concentration-dependent manner.

< Experimental Example  3> Investigation of the mechanism of action of cytokine production

<Experiment 3-1> NF -κB expression inhibitory activity- luciferase assay

Renilla is a recombinant vector and control reporter obtained by incubating HEK 293T cells in 24 well plates at 5X10 ⁴ / well and inserting the NF-κB gene into the pGL3-basic luciferase expression vector (Promega). pRL-SV-40 Pramide (Promega) with luciferase cDNA was co-transfected into HEK 293T cells using lipofectamine (Invitrogen). After 24 hours, the samples were treated and the cells were lysed to measure the activation of luminescent enzymes by luciferase assay system (Promega). Ketotifen was used as a positive control.

The results are shown in FIG. 10. All of the isolated effective substances have been shown to inhibit the expression of NF-κB above the positive control.

Experimental Example 3-2 MAPK and p- AKT Production Inhibitory Activity Test- western blot

RBL-2H3 cells of the above Experimental Example 2, which were activated with IgE and its antigen and treated with PBS, were washed with PBS, followed by lysis buffer (50 mM Tris-HCl, pH 8.0 with 150 mM sodium chloride, 1% NP40, 0.5% sodium). deoxycholate, 0.1% sodium dodecyl sμl). Protein was extracted and quantified by centrifugation at 13,000 rpm for 20 minutes. Quantified proteins were transferred to polyvinylidene difluoride membrane after electrophoresis using SDS-PAGE (sodium dodecyl sμlFate polyacrylamide gel electrophoresis). The membrane was blocked with phosphate buffered saline containing 0.1% Tween 20 and 5% skim dried milk. The membrane was blocked with phosphate buffered saline containing 0.1% Tween 20 and 5% skim dried milk. Membrane protein was reacted with primary antibody (1: 1000) (Cell signaling) and secondary antibody (1: 10000) (SantaCruz) combined with horseradish peroxidase, and then MAPK (ERK, JNK and p38) and p-AKT expression level was evaluated by comparing the expression level of β-actin.

The results are shown in [Fig. 11], all of the separated effective substances inhibit the expression of MAPK and p-AKT in a concentration-dependent manner, similar to the positive control (Ketotifen) at the same treatment concentration (300uM) It can be seen that the degree of activity.

< Experimental Example  4> Lipoxygenase  Activity inhibition experiment

20 μl of the sample was added to 2 ml of 0.1 M Tris buffer (pH 8.5), and 20 μl of the substrate linoleic acid (final concentration 110 uM) was mixed and reacted for 5 minutes at room temperature. 20 μl of soybean lipoxygenase (Type, 500 UNIT / final concentration) was added to the reaction solution, followed by reaction for 5 minutes, and absorbance at 234 nm was measured.

As a positive control, NDGA (nordihydroguaiaretic acid), a phenolic food antioxidant, was used, and the IC ₅₀ value of each sample was obtained and evaluated.

The results are shown in [Table 2].

Inhibition of lipoxygenase activity sample IC ₅₀ (uM) Compound 1 233.06 Compound 2 184.58 NDGA 67.46

<110> LEE, HAI-SOO <120> Composition for Improving Allergy Disease Using the Effective          Compound Which Cay Be Isolated from an Extract of Wheat Bran <130> PP10-000 <160> 12 <170> KopatentIn 2.0 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 tgtatgctac catctggctt cgg 23 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 gtttggaaca gtcgctcgtc atc 23 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 caccacgctc ttctgtctac tgaa 24 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 ccggactccg tgatgtctaa gtact 25 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 accttgcttc accctgttc 19 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 ttgtgagcgt ggactcattc 20 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 gcccttcagg aacagctatg a 21 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 tgtcaacaac atcagtccca aga 23 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 tagaagtgat gccccagg 18 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 tcatcctcca cctgctccac tgc 23 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 gagtcaacgg atttggtcgt 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 gacaagcttc ccgttctcag 20

Claims (10)

A composition for improving allergic diseases comprising 5-n-nonadecylresorcinol or 5-n-henecosilesinocinol as an active ingredient which can be isolated from wheat bran extract.
The method of claim 1,
The allergic diseases include contact dermatitis, atopic dermatitis, eczema, pruritus, hay fever, asthma, bronchitis, urticaria, vasculitis, rhinitis, gastroenteritis, diarrhea, interstitial pneumonia, arthritis, ophthalmitis, conjunctivitis, neuritis, otitis media , Granulomatosis, encephalomyelitis, cystitis, laryngitis, purpura (紫 물), food allergy, insect allergy, drug allergy, metal allergy and anaphylactic shock, allergic disease improver composition, characterized in that one selected from the group consisting of.
The method according to claim 1 or 2,
The composition is an allergic disease improving composition, characterized in that the pharmaceutical composition.
The method according to claim 1 or 2,
The composition is an allergic disease improving composition, characterized in that the food composition.
The method according to claim 1 or 2,
The composition is an inflammatory disease improving composition, characterized in that the cosmetic composition.
Inflammatory disease-improving agent composition comprising 5-n-nonadecylresorcinol or 5-n-henecosilesinocinol as an active ingredient which can be isolated from wheat bran extract.
The method of claim 6,
The inflammatory diseases include asthma, allergic and non-allergic rhinitis, chronic and acute rhinitis, chronic and acute gastritis or enteritis, ulcerative gastritis, acute and chronic nephritis, acute and chronic hepatitis, chronic obstructive pulmonary disease, pulmonary fibrosis, irritability Bowel Syndrome, Inflammatory Pain, Migraine, Headache, Back Pain, Fibromyalgia, Fascia Disease, Viral Infection, Bacterial Infection, Fungal Infection, Burn, Surgical or Dental Surgery, Prostaglandin E-Over Syndrome, Atherosclerosis Atherosclerosis, gout, arthritis, rheumatoid arthritis, ankylosing spondylitis, Hodgkin's disease, pancreatitis, conjunctivitis, irisitis, scleritis, uveitis, dermatitis, atopic dermatitis, eczema and multiple sclerosis Disease composition.
The method according to claim 6 or 7,
The composition is an inflammatory disease improving composition, characterized in that the pharmaceutical composition.
The method according to claim 6 or 7,
The composition is an inflammatory disease improving composition, characterized in that the food composition.
The method according to claim 6 or 7,
The composition is an inflammatory disease improving composition, characterized in that the cosmetic composition.

Images