KR101284365B1 - Composition for Improving Allergy Disease Using Wheat Bran - Google Patents

Abstract

The present invention relates to a composition for improving allergic diseases using wheat bran extract having an activity of inhibiting degranulation of mast cells and showing anti-allergic activity even in passive skin hypersensitivity animal experiments.

Description

Composition for improving allergic diseases using wheat bran extract {Composition for Improving Allergy Disease Using Wheat Bran}

The present invention relates to a composition for improving allergic diseases using 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.

Allergic reactions are mediated by mast cells, which are widely distributed in organs of the whole body such as the skin, respiratory organs, mucous membranes of the gastrointestinal tract, around lymphatic vessels, around blood vessels, and in the brain, and are known as cells causing allergic reactions.

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).

Specifically, when allergens invade a living body, allergens are first recognized by antigen-presenting cells, and these antigen-presenting cells differentiated 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.

The generated IgE binds to the high-affinity IgE receptor FcεRⅠ of mast cells, and then, when exposed to the same allergen, allergens bind to IgE bound to the mast cells' FcεRⅠ and cause cross-linking of these receptors. It acts as a series of signaling reactions.

Mast cells activated through this signaling reaction secrete histamine, proteoglycans, proteases, leukotrien, prostaglandin, and cytokines by degranulation or newly synthesized It mediates a series of allergic reactions, such as vasodilation, permeability hyperstimulation, nerve ending stimulation, mucus secretion, and smooth muscle contraction.

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.

Since the allergic reaction is caused by activation of mast cells such as degranulation of mast cells, a substance that inhibits activation of mast cells can be used as a therapeutic agent for allergic diseases (J. Korean Soc. Appl. Biol. Chem. 48 (4), 315-321 (2005)).

The present invention is completed by confirming that wheat bran extract inhibits the activation of mast cells.

It is an object of the present invention to provide an ameliorative agent composition for allergic diseases.

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

The inventors of the present invention, as confirmed in the following Examples and Experimental Examples, after treatment with mast cell line RBL-2H3 (rat basophilic leukemia) activated with IgE and antigen (DNP-BSA), beta- hexosamini It was confirmed that the production of the enzyme (β-hexosaminidase) is inhibited. 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)).

In particular, wheat bran extract showed anti-allergic activity in a dose-dependent manner in passive cutaneous anaphylaxis (PCA) animal experiments, which are frequently used for the development of allergic drugs.

The allergic disease improver composition of the present invention is completed based on the results of these experiments, and is characterized by including wheat bran extract as an active ingredient.

In the present specification, the "branch" means a by-product obtained in the process of milling wheat. These by-products usually consist of the rind and the embryo, except for the endosperm, which becomes flour.

In the present specification, regardless of the extraction method, the "extract" is obtained by immersing the wheat bran to be extracted by immersing it in water containing distilled water, lower alcohols having 1 to 4 carbon atoms such as methanol, ethanol, butanol, or a mixed solvent thereof. Crude extracts and their crude extracts are understood as meaning including extracts fractionated with the solvents listed above. Regardless of the extraction method, as long as the extraction object is extracted through the step of immersing in the extraction solvent, the extraction method may be any method such as cooling, refluxing, heating, ultrasonic radiation, and the like. In the case of the fractionated extract, the crude extract is suspended in a specific solvent and mixed and fixed with a solvent of different polarity. The crude extract is adsorbed onto a column filled with hydrophobic resin, and the fraction obtained by using the solvent as a mobile phase is obtained. It is meant to include. Hydrophobic resins that can be used can be appropriately selected from among commercially available products such as Amberlite XAD-2 ™, Diaion HP-20 ™ used in the examples below. The meaning of the extract includes a concentrated liquid extract or solid extract from which the extraction solvent is removed.

In the present specification, the above-mentioned "active ingredient" means an ingredient that exhibits the desired activity alone or can exhibit activity together with a carrier that is itself inactive.

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.

Meanwhile, the composition of the present invention may include wheat bran extract as an active ingredient in any amount (effective amount) as long as it can exhibit improvement activity of allergic diseases intended to be treated according to use, formulation, formulation purpose, etc. Effective amounts 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 wheat bran extract as an active ingredient.

IgE and its antigen-activated mast cells not only secrete histamine, which causes allergic reactions by degranulation, but also TNF-α, IL-1β, IL-4, IL-5, IL-6, IL-13, Cytokines such as INF-γ are synthesized and secreted. TNF-α, IL-1β, IL-6 and the like are representative inflammatory cytokines. This inflammatory cytokine induces the expression of iNOS, which produces NO that causes cytotoxicity and various inflammatory responses, and the expression of COX-2, which is 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).

The following experimental example shows that wheat bran extract not only inhibits the production of inflammatory cytokines in activated mast cells but also inhibits iNOS expression and COX-2 expression. In addition, the inhibitory activity of NF-κB, an inflammatory cytokine transcription factor, and the inhibitory activity of mitogen-activated protein kinase (MAPK) (ERK, JNK and p-38), which are involved in the activation of NF-κB, are shown.

These experimental results show that wheat bran extract can be usefully used as an inflammatory disease ameliorator.

As used herein, the term "inflammatory disease" may be defined as any condition specified as 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 reaction is enzyme secretion (e.g., iNOS, COX-2, etc.) activation, release of inflammatory mediators (e. G., NO, TNF-α, IL -6, IL-1β, PGE 2 related to various inflammatory mediators and the immune cells ), Bodily fluid infiltration, cell migration, and tissue destruction, and manifest externally by symptoms such as erythema, pain, edema, fever, deterioration or loss of specific function 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 improving composition of the inflammatory disease of the present invention, the meaning, effective amount, etc. of the extract of wheat bran is effective as it is described with respect to the improving composition of the allergic disease of the present invention.

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), in addition to bran extract, which is an active ingredient, in addition to pharmaceutically acceptable carriers, excipients, and the like. Aqueous or oily suspensions for injection), 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 route of administration, age, sex, weight, and patient's severity of the patient, the dose is limited in any aspect to the scope of the present invention Should not be understood to be.

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

The food composition of the present invention can be produced as a health supplement food, a special nutrition supplement food, a functional beverage and the like.

The food composition of the present invention may contain sweetening agents, flavoring agents, physiologically active ingredients, minerals and the like in addition to the active ingredients thereof.

Sweetening agents may be used in an amount that sweetens the food in a suitable manner, 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.

Flavors may be used to enhance taste or flavor, 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. Synthetic flavors may be used depending on the case, and synthetic flavors such as esters, alcohols, aldehydes, terpenes and the like may be used.

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

In addition, the food composition of the present invention may contain preservatives, emulsifiers, acidifiers, thickeners and the like as needed in addition to the above sweeteners.

Such preservatives, emulsifiers and the like are preferably added in a very small amount as long as they can attain an application to which they are added. The term " trace amount " means, when expressed numerically, in the range of 0.0005% by weight to about 0.5% by weight based on the total weight of the food composition.

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

Examples of the emulsifier which can be used include acacia gum, carboxymethyl cellulose, xanthan gum, pectin and the like.

Examples of the acidulant that can be used include acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, and phosphoric acid. Such an acidulant may be added so that the food composition has a proper acidity for the purpose of inhibiting the growth of microorganisms other than the purpose of enhancing the taste.

Agents that may be used include suspending agents, sedimentation agents, gel formers, bulking agents and the like.

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 improving composition and the inflammatory disease improving composition of the present invention can be commercialized as a drug or functional food.

1 is a result showing the degranulation inhibitory activity of mast cells of wheat bran extract.
Figure 2 is a result showing the anti-allergic activity in animal experiments of wheat bran extract.
Figure 3 is a result showing the cytokine production inhibitory activity in mast cells of wheat bran extract.
Figure 4 shows that wheat bran extract inhibits the expression of NF-κB transcription factor of cytokines.
5 is a result showing that the wheat bran extract MAPK expression inhibitory activity that regulates the activation of NF-κB.

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  Preparation of extract

200 g of Red-dog was placed in 2 L of water and extracted with hot water for 6 hours. The hot water extract was filtered and the filtrate was placed in a column filled with Diaion HP-20 resin, and 100% ethanol and water were used as the mobile phase, and the sample passed through 100% ethanol was called Red-dog A. Red-dog B was used for the experiment.

< Experimental Example > Anti-allergy  Activity and anti-inflammatory activity experiment

< Experimental Example  1> Mast cell Degranulation  Inhibitory activity Experiment - β- Hexosaminidase assay

RBL-2H3 cell line, a rat basophlic leukemia cell line, was incubated with MEM medium containing 15% FBS, 100unit / ml penicillin and streptomycin for 24 hours at 2X10 ⁵ / well at 37 ° C and 5% CO _2. .

After removing the supernatant, MEM medium containing 25 ng / ml anti-DNP IgE was added and incubated for 4 hours. After incubation, washed with PIPES buffer (25mM PIPES, 119mM NaCl, 5mM KCl, 1mM CaCl ₂ , 0.4mM MgCl ₂ , 40mM NaOH, 5.6mM glucose, 0.1% BSA) and incubated for 10 minutes. After pre-culture, the sample of Example was treated for 20 minutes at a concentration of 25, 50, 100 μg / ml, and reacted for 20 minutes at 50 ng / ml DNP-BSA. After the reaction was terminated at 4 ° C., 25 μl of the supernatant was put in a 96 well plate, and 25 μl of 1 mM P-nitrophenyl-acetyl-β-D-glucosamide was added and reacted at 37 ° C. for 1 hour. 0.1M citrate buffer was added to terminate the reaction, and the absorbance was measured with an ELISA reader at 405 nm wavelength. Ketotifen (100 μg / ml) was used as a positive control.

The results are shown in FIG. 1, and all of the samples of Example showed inhibition of degranulation in a concentration-dependent manner, and showed similar activity to ketotifen, a positive control group, at the same treatment concentration.

<Experiment 2>, wherein the allergy animal model using experimental allergic activity - Passive Cutaneous and minjeung (PCA) test

Passive skin allergic reaction was performed by intradermal injection of anti-DNP IgE (0.5 μg / site) into ICR mouse sensitized skin 48 hours after oral administration of the sample (Red-dog A) of the example and the control ketotifen (100 mg / kg) was administered intraperitoneally. After 1 hour, a 1: 1 mixture of 4% Evans blue and DNP-BSA was injected through the tail vein of the rat. After 1 hour, the experimental animals were photographed and shown in FIG. 2. Referring to FIG. 2, it can be seen that the amount of pigment reacting with the substance causing allergy decreases with the naked eye according to the dose of the sample of the example.

< Experimental Example  3> Inhibitory Activity of Cytokine Production- RT PCR

RBL-2H3 cells were incubated in 6 well plates at 1 × 10 ⁶ / well and then 25 ng / ml IgE was added for 4 hours. Thereafter, 50 ng / ml DNP-BSA was added after the sample treatment in Example to react at 37 ° C. for 20 minutes. After washing twice with PBS, Trizol (Invitrogen) and chloroform (Sigma) were added and reacted to separate RNA. After quantification using 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 2] below, and the control gene was GAPDH. Ketotifen (100 μg / ml) was used as a positive control.

primer rTNF-α F: 5 'CAC CAC GCT CTT CTG TCT ACT GAA C 3' (SEQ ID NO: 1) R: 5 'CCG GAC TCC GTG ATG TCT AAG TAC T 3' (SEQ ID NO: 2) rIL-1b F: 5 'CAC CTC TCA AGC AGA GCA CAG 3' (SEQ ID NO: 3) R: 5 'GGG TTC CAT GGT GAA GTC AAC 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-5 F: 5 'TGA GCA CAG TGG TGA AAG AGA C 3' (SEQ ID NO: 7) R: 5 'AAT CCA GGA AAT GCC TGG TC 3' (SEQ ID NO: 8) rIL-6 F: 5 'GCC CTT CAG GAA CAG CTA TGA 3' (SEQ ID NO: 9) R: 5 'TGT CAA CAA CAT CAG TCC CAA GA 3' (SEQ ID NO: 10) rIL-10 F: 5 'TAG AAG TGA TGC CCC AGG 3' (SEQ ID NO: 11) R: 5 'TCA TCC TCC ACC TGC TCC ACT GC 3' (SEQ ID NO: 12) rIL-11 F: 5 'TTG GTA CTT GGA GCG G 3' (SEQ ID NO: 13) R: 5 'CAC TGT GAA TAG ACT TCG T 3' (SEQ ID NO: 14) rIL-13 F: 5 'ACA GCT CCC TGG TTC TCT CA 3' (SEQ ID NO: 15) R: 5 'CCC CCA TTC ACT ACA CAT CA 3' (SEQ ID NO: 16) rIFN-γ F: 5 'AGG ACG GTA ACA CGA AA 3' (SEQ ID NO: 17) R: 5 'CTG TGG TTG TTC ACC TC 3' (SEQ ID NO: 18) rCOX-2 F: 5 'TGT ATG CTA CCA TCT GGC TTC GG 3' (SEQ ID NO: 19) R: 5 'GTT TGG AAC AGT CGC TCG TCA TC 3' (SEQ ID NO: 20) rNOS F: 5 'CAT TGG AAG TGA AGC GTT TCG 3' (SEQ ID NO: 21) R: 5 'CAG CTG GGC TGT ACA AAC CTT 3' (SEQ ID NO: 22) GAPDH F- GAGTCAACGGATTTGGTCGT (SEQ ID NO: 23) R-GACAAGCTTCCCGTTCTCAG (SEQ ID NO: 24)

The results are shown in FIG. 3. Referring to FIG. 3, it can be seen that the sample of the example inhibits the production of cytokines in a concentration-dependent manner. When the treatment concentrations are the same, the sample shows similar activity to that of the positive control group (PC).

< Experimental Example  4> Investigation of the mechanism of action of cytokine production

Experimental Example 4-1 NF- κB Expression Inhibitory Activity Experiment- 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 of the Example were treated, and the cells were lysed to measure the activation of the luminase by luciferase assay system (Promega). Ketotifen (100 μg / ml) was used as a positive control.

The results are shown in FIG. 4. All of the samples in the example showed that the concentration-dependent inhibition of the expression of NF-κB.

Experimental Example 4-2 MAPK Production Inhibition Activity Experiment- western blot

RBL-2H3 cells of Example 3, which were activated with IgE and its antigen and treated with the sample of Example, 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 13000 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 proteins were reacted with primary antibody (1: 1000) (Cell signaling) and secondary antibody (1: 10000) (SantaCruz) complexed with horseradish peroxidase, followed by ECL western detection reagent (Thermo scientific). The expression level of JNK and p38) was evaluated by comparison with the expression level of β-actin.

The results are shown in <Figure 5>, it can be seen that all the samples of the example inhibit the expression of MAPK in a concentration-dependent manner.

Claims (5)

delete delete delete Contains wheat bran extract as an active ingredient,
The wheat bran extract is antihistamine composition, characterized in that (I) or (II) below.
(I) an extract obtained by putting hydrothermal extract of wheat bran into a column filled with hydrophobic resin and using 100% ethanol as a mobile phase; And
(Ⅱ) The extract obtained by putting the hydrothermal extract of wheat bran into a column filled with hydrophobic resin and using water as a mobile phase
5. The method of claim 4,
The composition is an antihistamine composition, characterized in that the pharmaceutical composition.

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