KR20190071984A - Composition for alleviating allergic disease - Google Patents

Abstract

The present invention relates to a composition for alleviating allergic diseases, which contains arctigenin and tannic acid as active components, has an anti-inflammatory effect, and is excellent in inhibiting histamine release, thereby having an alleviation effect on allergic diseases such as rhinitis, asthma, atopy and the like, wherein the composition contains arctigenin and tannic acid as active components.

Description

[Composition for alleviating allergic disease]

The present invention relates to a composition for alleviating allergic diseases, and more particularly, to a composition for relieving allergic diseases such as rhinitis, asthma, and atopy, which is excellent in antiinflammatory effect and inhibition of histamine release and contains actinin and tannic acid as effective ingredients. To a composition for relieving allergic diseases.

Allergic symptoms are caused when exogenous allergen is introduced into the body, IgE antibody against the antigen is produced in the immune cells, and IgE is bound to mast cells and platelet IgE receptors, Is associated with IgE, it is known that many substances such as histamine and serotonin are released when mast cells or platelets are degranulated.

A typical example of an allergic symptom is a skin-related abnormality that is accompanied by symptoms that can be referred to as a series of atopic diseases such as dry skin, keratinization, itching, inflammation due to secondary microbial infection, and persistent and repeated hypersensitivity . Allergic symptoms of elderly people, children, and infants are characterized by chronic diseases caused by repeated exposure to external environmental factors. The major cause of skin related abnormalities is due to various allergens caused by the urbanization and industrialization of the living environment, and recently, it occurs in various forms regardless of seasonal characteristics.

Therefore, various forms of drugs and preparations have been developed depending on the therapeutic purpose such as a moisturizer, an antibacterial agent, an anti-inflammatory agent, a steroid hormone agent, but repeated use for a long period of time is inevitable.

Forms that are mainly used for managing skin related abnormalities include emollients that form a thin film on the skin to maintain the moisture and flexibility of the skin, external-use ointments of hydrocortisone, antihistamines, steroids or antihistamines A bandage containing an oil component for improving dryness, an oil component external agent for improving dryness, a diet, and the like.

Korean Patent Laid-Open Publication No. 1992-0021158 (title of the invention: composition for prevention and treatment of allergic diseases containing extract of mulberry bark extract) discloses a composition for the prevention and treatment of allergic diseases containing extract of mulberry bark extract.

Korean Patent Registration No. 10-0821926 entitled "Anti-mite allergenic neutralizing agent including plant extract and composition comprising the same" relates to a mite allergen neutralizer comprising a plant extract and a composition comprising the same A natural plant extract and a plant essential oil which can neutralize allergen causing all kinds of diseases caused by main house dust mite species all over the world and one or more kinds of extracts of active substances contained therein or A protein neutralizing composition containing essential oils and a production method thereof ".

Korean Patent Laid-Open Publication No. 10-2010-0087785 (the name of the invention: a skin disease-inhibiting composition using a linkage and a method for producing the same), the present invention relates to an extract of a plant extract useful in the treatment of skin diseases, Which is useful for prevention and treatment of skin diseases such as atopy, psoriasis and the like, by confirming that antioxidant activity and epidermal growth inhibition effect of antioxidative effect of antioxidative effect is excellent. Skin-related diseases and detoxifying action. Therefore, the present invention is an excellent invention useful for the prevention and treatment of skin diseases such as atopy and psoriasis. "

Korean Patent Registration No. 10-0566542 entitled " Health food composition for prevention and treatment of allergic rhinitis and cold and method for producing the same ", the present invention relates to a composition for prevention and treatment of allergic rhinitis, The present invention relates to a health food composition for prevention and treatment of allergic rhinitis and colds containing as an active ingredient an active ingredient, a yam, a yam, a yam, a yam, a gold, a mint, and a tea.

However, there is still a desperate need for the development of natural materials that can improve and prevent allergic symptoms and reduce side effects in drug treatment.

The present inventors have conducted intensive studies to develop a composition that maximizes the alleviating effect of allergic diseases by selecting natural materials that have an anti-inflammatory effect to fundamentally improve allergic diseases suffered by modern humans and that help inhibit histamine release. As a result, The present inventors have developed a composition for alleviating allergic diseases which has an effect of suppressing allergic diseases such as rhinitis, asthma and atopy, which is excellent in antiinflammatory effect and inhibition of histamine release. ( in vitro ) and in vivo experiments to complete the present invention.

The composition for relieving allergic diseases according to the present invention comprises actinin and tannic acid as active ingredients.

According to the present invention, it contains actinin and tannic acid as active ingredients, and is excellent in anti-inflammatory effect and inhibition of histamine release, thereby providing a mitigating effect of allergic diseases such as rhinitis, asthma, and atopy.

FIG. 1 is a graph showing comparison of inhibition of histamine release of natural product extracts.
Figure 2 is a graph showing histamine release inhibition comparisons of burdock extracts.
3 is a graph showing the comparison of COX-2 inhibitory activity with tannin acid concentration.
Figure 4 is a graph showing the inhibition of histamine release by 95% actinogen (as a concentrated burdock extract concentrate) and 98% tannic acid mixture.
5 is a graph showing the effect of 5-lipoxygenase inhibition of AT101 according to the present invention.
6 is a graph showing the inhibitory effect of AT101 according to the present invention on the induction type NO synthase (iNOS).
7 is a graph showing the COX-2 inhibitory effect of AT101 according to the present invention.
Figure 8 is a graph showing the results of MTT analysis of 95% actinin (as a burdock extract tablet concentrate).
9 is a graph showing the results of 98% tannic acid MTT analysis.
10 is a graph showing histamine-inhibiting activity.
11 is a graph showing the effect of inhibiting TNF-a expression.
12 is a graph showing the IL-6 expression inhibitory effect.
13 is a graph showing the effect of inhibiting evans blue leakage.
Figure 14 is a photograph of Evans blue leakage inhibition.
15 is a graph showing the effect of inhibiting leukotrienes in serum.
16 is a graph showing the effect of inhibiting prostaglandin E ₂ in serum.
17 is a graph showing the visual score of the DNCB animal test.
18 is a graph showing the results of changes in ear thickness in ear swelling of the animal experiment with DNCB.
19 is a graph showing the results of spleen weight change in the DNCB animal experiment.
FIG. 20 is a graph showing the results of DNCB animal experiment ear weight change. FIG.
FIG. 21 is a graph showing the results of leukocyte count changes during blood analysis of animal experiments in DNCB. FIG.
FIG. 22 is a graph showing the eosinophil number change in the blood analysis of the DNCB animal experiment. FIG.
FIG. 23 is a graph showing the results of changes in the number of basophils during blood analysis of animal experiments in DNCB. FIG.
24 is a graph showing the results of TNF-a changes in the tissue analysis of DNCB animal experiment ELISA.
25 is a graph showing the results of IFN-y changes in an ELISA tissue analysis of DNCB animal experiments.
FIG. 26 is a graph showing the results of IL-17 change during ELISA tissue analysis of DNCB animal experiment. FIG.

Hereinafter, the present invention will be described in detail with reference to specific examples.

The composition for relieving allergic diseases according to the present invention is characterized by containing actinin and tannic acid as active ingredients. Hereinafter, the composition according to the present invention comprising actinin and tannic acid as active ingredients is abbreviated as AT101.

In the drawings, the symbols are in accordance with the legend described in the following [description of the symbols] unless otherwise specified, and the numbers in parentheses in the symbols indicate addition amounts (ppm) in the case of Figures 10 to 12, And in the case of Figures 13 to 26, the dose (unit mg / kg).

The actinine and tannic acid constituting the composition according to the present invention can be obtained as follows.

Actinogen can be obtained from extracts of various plants, in particular from extracts such as Burdock, Associate, Mantum, Forsythia, Hymenoptera, Casualty, Rockfall, etc. Hereinafter, the description will be made on the basis of the burdock extract as an example.

The burdock extract is obtained by washing the burdock root with water, ethanol, or a mixed solvent thereof, preferably water, at 20 to 100 ° C, preferably in the range of 2 to 20 times the weight of the raw material, An extraction method such as hot water extraction, ultrasonic extraction, reflux cooling extraction and the like at an extraction temperature of 50 to 95 ° C for 1 hour to 2 days, preferably 2 to 8 hours, 1 to 10 times, preferably 2 to 5 times , Preferably by performing hot water extraction.

Burdock (Arctium lappa L. ) is a plant of the plant family, the plant of the gentian plant, the plant of the dicotyledonous plant, the plant of the fir tree, and the flower of asteraceae. The varieties have long roots, thick agar, good quality meat, and short roots. The roots contain inulin and some palmitic acid. In Europe, it is used as a diuretic and sweating agent. Seeds are used as diuretics when there is a swelling, and as an antidote to sore throats and poisonous insects. It grows much in Japan and is wild in Europe, Siberia and northeastern China. In addition, in one room, seed is used as a medicinal product as evil spirit. Pharmacological effect is the effect of scavenging the air (heat wave), and it has the effect of detoxification, the exorcism, and the detoxifying effect, so it can be used for the fever seawater (wind heat cough), sore throat (rubella), rubella Is used. The obtained burdock extract is filtered and concentrated under reduced pressure or concentrated in vacuo to obtain an extract concentrate. In addition, n-hexane can be used for degreasing and the use of Diaion HP-20 ionic resin or Sephadex LH-20 column is effective It is possible to increase the extraction yield of the component actinin.

In the case of tannic acid, the anti-inflammatory effect has already been proven in many literatures, and the purified extract having a content of 95% or more can be easily purchased and used.

In addition, the raw materials have been used as food for a long time, and the composition of the present invention extracted therefrom also has no toxicity and side effects.

Specifically, the composition for alleviating allergic diseases according to the present invention comprises 98% tannic acid in a weight ratio of 95% actinin (as a concentrated burial-rinse concentrate) to 2 to 10: 1, preferably 4 to 8: 1, Is 5: 1.

In addition, the present invention provides a method for preparing an allergic disease-mitigating composition comprising the steps of: (1) obtaining a burdock extract from burdock wastes; And (2) actinin and tannic acid in a weight ratio of 95% actinin (as a burdock extract tablet concentrate): 98% tannic acid in a weight ratio of 2 to 10: 1, preferably 4 to 6: 1, more preferably 5: And mixing the resultant mixture.

5-lipoxygenase inhibitory effect, inducible nitric oxide synthase (iNOS) inhibitory effect, COX-2 (cyclooxygenase-2) inhibitory effect of the composition obtained by the above- 2) inhibitory effect, histamine-free inhibitory effect, and inflammatory cytokine (TNF-α, IL-1β, IL-6) In addition, in order to investigate the effect of alleviating allergic diseases, animal experiments were carried out and it was confirmed that they exerted an excellent effect in alleviating allergic diseases.

The composition for relieving allergic diseases according to the present invention can be commercialized in various forms. Examples of commercialization include, for example, a health functional food for alleviating allergic diseases, a coating agent for alleviating allergic diseases, a general food for alleviating allergic diseases, etc., containing the above composition as an active ingredient, It is to be understood that the invention is not intended to be limited thereto. These may further comprise suitable carriers, excipients and diluents conventionally used in the preparation of natural product compositions.

In addition, the composition for relieving allergic diseases according to the present invention may be formulated into various forms. Examples of formulations include, for example, tablets, capsules, pills, liquids and the like, but it should be understood that the invention is not intended to be limited thereto.

The composition according to the present invention may be formulated into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions and syrups according to a conventional method. Examples of carriers, excipients and diluents that can be contained in the composition comprising the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate , Calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, honey, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate and mineral oil. When formulating the composition, it is prepared using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are 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 or lactose, gelatin, . In addition to simple excipients, lubricants such as magnesium stearate 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 .

The preferred dosage of the composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the composition of the present invention is preferably administered at 0.1 to 1 g per day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

Since the composition of the present invention has no toxicity and side effects, it can be safely used for prolonged use for preventive purposes.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

It should be noted, however, that the following examples and experimental examples are illustrative of the present invention and that the present invention is not limited to the following examples and experimental examples.

Test Example  1: Selection of natural products and preparation of extracts

There are 20 kinds of natural products that are anticipated to have antiallergic effect through literature review (cinnamon, alfalfa, chrysanthemum, gigyeong, canary, Rocks, guava, white paper, evening primrose, aloe) were selected and purchased from the raw material supplier. To prepare a natural product extract, 100 g of the powder obtained by drying each 1 kg of the natural product, and then grinding it, was taken in a house vat, and then extracted with 2 times repeatedly at 80 ° C for 4 hours in an 80%

Experimental Example  1: Comparison of histamine release inhibition

SD rats at approximately 6 weeks of age, which had been subjected to a weekly purifying period, were sacrificed and used in the experiment.

30 mL of HEPES-DMEM (HEPES-Dulbecco's modied Eagle's medium) was injected into the abdominal cavity of the sacrificed rats, followed by 3 minutes of abdominal massage. HEPES-DMEM in which mast cells were suspended in the peritoneal cavity was taken using a 10 mL syringe and washed three times with clean medium. The number of mast cells was measured by a microscope using a hemacytometer, and the cells were dispensed into a 24-well cell culture plate at 5 × 10 ⁵ / well. The treated cells were treated with 10 ppm of the sample except for the normal (Normal) and the control (Control), and cultured at 37 ° C and 5% CO ₂ for 10 minutes. Subsequently, Compound 48/80 was treated at a concentration of 5 μg / ml in all wells except for the normal group, and cultured at 37 ° C and 5% CO ₂ for 20 minutes. The supernatant of the cell culture was quantitated and used for the experiment.

Histamine was measured using the Histamine ELISA Kit Instructions (NEOGEN com, USA). The cell culture supernatant was diluted to a final protein amount of 1 mu g per well in a 96-well plate, and 50 [mu] l of each solution was dispensed. Enzyme conjugate (50 μl) was added to all wells, reacted at room temperature for 45 minutes, and washed three times with 200 μl of washing buffer. Thereafter, 150 쨉 l of K-blue substrate is dispensed into all wells, reacted at room temperature for 30 minutes, and absorbance is measured at 650 nm with an ELISA reader. The amount of histamine was quantitated using a standard curve (0, 2.5, 5, 10, 20, 50 ng / 50 μl) and the results are shown in FIG.

As shown in FIG. 1, the natural product extracts of eight species (yeast, forsythia, burdock, mangmundong, whitehead, casual, Was the most excellent.

Experimental Example  2: HPLC  analysis - Actinine  content

To investigate the content of actinogen in 8 kinds of natural extracts with excellent histamine release inhibition effect (yeast, forsythia, burdock, mungmundong, whitehead, casualty, RP-HPLC was performed using Model code 6CE (Waters, Milford, Mass.). Waters 717 _{plus for} auto sampler, Inline Degasser-AF for degassing unit, Luna C18 Column (250 × 4.6 mm, particle size 5 μm, manufactured by Phenomenex Co., Ltd.) ) Were used. Actinine was analyzed by methanol and tertiary distilled water at 55:45 (v / v), filtered through a 0.45 μm Millipore Durapore filter before use, and deaerated by sonication. The RP-HPLC elution was carried out by the isocratic elution method at a flow rate of 1.0 ml / min, a sample injection volume of 10 μl, and a column temperature of 30 ° C. for 35 min at 280 nm. 0.2 탆), and the results are shown in Table 1 (HPLC analysis - content of actinogen).

Natural extract Actinin content (%) Abalone 0.284 forsythia 0.262 burdock 0.315 McMundong 0.226 Bae Hoon 0.194 Casualty 0.246 Seven leaf fence 0.252 Rockfall etc. 0.217

As shown in Table 1, as shown in Table 1, all of the natural products of eight (yeonjong, forsythia, burdock, mangmundong, The highest content of actinine in the burdock extract was 0.315%.

Example  1: Purification of burdock extract - Actinine

In order to increase the content of actinine in the burdock, which is the active ingredient of the extract, the same amount of n-hexane was added thereto, followed by degreasing and concentration to obtain 20 g of a powder. The powder was dissolved in 1 liter of 10% MeOH, and pectinase and cellulase (cellulase) were added, and the mixture was hydrolyzed at 50 ° C for 12 hours with stirring. The hydrolyzed extract was passed through a Diaion HP-20 ionic resin column, eluted with 80% MeOH solvent, passed through a Sephadex LH-20 column, and the fraction containing the active ingredient was selected and purified by a vacuum rotary evaporator at 40 ° C , And then dried to obtain burdock extract (actinin 43.24%)

Purified burdock extract (95% of actinogen) was also purchased.

Experimental Example 3: 95% Of Actinine (as Burdock Extract Tablet Concentrate)  Comparison of histamine release inhibitory effect

SD rats at approximately 6 weeks of age, which had been subjected to a weekly purifying period, were sacrificed and used in the experiment.

30 mL of HEPES-DMEM was injected into the abdominal cavity of the sacrificed rats, and the abdominal massage was performed for 3 minutes. HEPES-DMEM in which mast cells were suspended in the peritoneal cavity was taken using a 10 mL syringe and washed three times with clean medium. The number of mast cells was measured by a microscope using a hemocyte counter, and the cells were dispensed into a 24-well cell culture plate at 5 × 10 ⁵ / well. The treated cells were treated with 10 ppm of the sample except for the normal group and the control group, and cultured at 37 ° C and 5% CO ₂ for 10 minutes. Subsequently, Compound 48/80 was treated at a concentration of 5 μg / ml in all wells except for the normal group, and cultured at 37 ° C and 5% CO ₂ for 20 minutes. The supernatant of the cell culture was quantitated and used for the experiment.

Histamine was measured using the Histamine ELISA Kit Instructions (NEOGEN com, USA). The cell culture supernatant per well was diluted to a final protein amount of 1 μg in a 96-well plate, and 50 μl of each was diluted. Enzyme conjugate (50 μl) was added to all wells, reacted at room temperature for 45 minutes, and washed three times with 200 μl of washing buffer. Thereafter, 150 쨉 l of K-blue substrate was dispensed into all wells, reacted at room temperature for 30 minutes, and absorbance was measured at 650 nm with an ELISA reader. The amount of histamine was quantified using a standard curve (0, 2.5, 5, 10, 20, 50 ng / 50 μl) and the results are shown in FIG. 2 (the parenthesized value in FIG. 2 is the content of actinogen).

As shown in FIG. 2, the inhibitory effect of histamine on the release of histamine was better when the content of actinogen in burdock extract was higher.

Experimental Example  4: Tannic acid  Comparison of anti-inflammatory effects by concentration - Measurement of COX-2 inhibitory activity

The purified tannic acid was purchased at three concentrations (40%, 70%, and 98%) to compare COX-2 inhibitory activity. COX Colorimetric Inhibitor Screening Assay Kit (Creative Biomart Com, USA) was used. 10 μl of EIA buffer solution, 10 μl of COX-2 enzyme and 10 μl of Heme were added to a 96-well plate coated with prostaglandin antibody, 10 μl of buffer solution was added to the well of the well, and 10 μl of the sample well was added so that the final concentration of the sample became 10 ppm Thereafter, reaction was carried out at 25 DEG C for 5 minutes. The colorimetric substrate and the substrate arachidonic acid were added thereto, and the absorbance was measured at a wavelength of 590 nm after 2 minutes. The degree of inhibition of COX-2 activity was calculated and compared with the calculation method (activity inhibition rate (%) = (1 - sample absorbance / control absorbance) X 100). The IC ₅₀ values are shown in Figure 3 in ppm, the minimum concentration needed to inhibit 50% of the generated enzyme.

As shown in FIG. 3, the higher the tannic acid content, the better the COX-2 inhibitory activity.

Example  2: The maximum inhibitory effect of histamine is 95% Actinine (burdock extract refine As a concentrate ) And 98% Tannic acid  Investigation of compounding ratio

SD rats at approximately 6 weeks of age, which had been subjected to a weekly purifying period, were sacrificed and used in the experiment.

30 mL of HEPES-DMEM was injected into the abdominal cavity of the sacrificed rats, and the abdominal massage was performed for 3 minutes. HEPES-DMEM in which mast cells were suspended in the peritoneal cavity was taken using a 10 mL syringe and washed three times with clean medium. The number of mast cells was measured by a microscope using a hemocyte counter, and the cells were dispensed into a 24-well cell culture plate at 5 × 10 ⁵ / well. The treated cells were treated with 10 ppm of the sample except for the normal group and the control group, and cultured at 37 ° C and 5% CO ₂ for 10 minutes. Subsequently, Compound 48/80 was treated at a concentration of 5 μg / ml in all wells except for the normal group, and cultured at 37 ° C and 5% CO ₂ for 20 minutes. Then, the protein culture supernatant was used for the experiment .

Histamine was measured using the Histamine ELISA Kit Instructions (NEOGEN com, USA). The cell culture supernatant per well was diluted to a final protein amount of 1 mu g in a 96-well plate, and 50 [mu] l of each was diluted. Enzyme conjugate (50 μl) was added to all wells, reacted at room temperature for 45 minutes, and washed three times with 200 μl of washing buffer. Thereafter, 150 쨉 l of K-blue substrate was dispensed into all wells, reacted at room temperature for 30 minutes, and absorbance was measured at 650 nm with an ELISA reader. The amount of histamine was quantified using a standard curve (0, 2.5, 5, 10, 20, 50 ng / 50 μl) and the results are shown in FIG.

As shown in FIG. 4, the inhibitory effect of histamine was shown in FIG. 4, in which 95% of actininin and 98% of tannic acid were actininin (as a concentrate of burdock extract): 98% tannic acid = 5: 1 Quot; AT101 ") is considered to be advantageous from the functional point of view.

Experimental Example 5: 5 - Lipoxygenase  Inhibition effect measurement

Lipoxygenase Inhibitor Screening Assay Kit (Cayman Chemical Com, USA) was used. To a 96-well plate, 90 μl of LOs (220 units / ml) enzyme and 10 μl of arachidonic acid substrate were added, and 10 μl of the sample was added per concentration. After reacting at room temperature for 10 minutes, 100 μl of chromogen After incubation at room temperature for 5 minutes, the absorbance at 490 nm was measured with an ELISA reader. The degree of inhibition of 5-lipoxygenase activity was calculated according to the following formula (activity inhibition rate (%) = (1 - absorbance of sample addition group / absorbance of sample addition group) X 100). The IC ₅₀ values are shown in Table 2 and FIG. 5 in ppm in terms of the minimum concentration required to inhibit 50% of the generated enzyme.

Sample 5-lipoxygenase inhibitory effect IC ₅₀ (ppm) Burdock extract
(95% of actinogen) 1775.18 98% tannic acid 0.81 AT101 0.77

5-Lipoxygenase Inhibition Activity As shown in Table 2 and FIG. 5, 98% tannic acid exhibited 50% inhibitory activity at 0.81 ppm, and compound AT101 showed 50% inhibitory activity at 0.77 ppm.

Experimental Example  6: Inducible NO synthase ( iNOS : inducible Nitric oxide synthase ) Inhibition effect measurement

Nitric oxide synthase inhibitor screening assay kit (Bioassay systems Com, USA) was used. 10 μl of NOS (12.5 unit / ml) enzyme and 25 μl of buffer were added to a 96-well plate. Then, 5 μl of the sample was added at a concentration of 5 μl and reacted at 25 ° C for 15 minutes. After completion of the reaction, 10 아 of arachidonic acid substrate and 0.5 G of GDH (glucose dehydrogenase) were added, followed by reaction at 37 캜 for 60 minutes. Subsequently, 200 쨉 l of chromogen was dispensed, followed by incubation at 37 캜 for 60 minutes After the reaction, the absorbance was measured at 540 nm with an ELISA reader. The degree of inhibition of NOS activity was calculated by the calculation method (activity inhibition rate (%) = (1 - absorbance of sample added / absorbance of sample not added) X 100). The IC ₅₀ values are shown in Table 3 and FIG. 6 in ppm in the minimum concentration required to inhibit 50% of the generated enzyme.

Sample iNOS inhibitory activity IC ₅₀ (ppm) Burdock extract
(95% of actinogen) 1083.59 98% tannic acid 4.61 AT101 4.21

As shown in Table 3 and FIG. 6, 98% tannic acid showed 50% inhibitory activity at 4.61 ppm and compound AT101 showed 50% inhibitory activity at 4.21 ppm.

Experimental Example  7: Measurement of COX-2 inhibitory effect

COX Colorimetric Inhibitor Screening Assay Kit (Creative Biomart Com, USA) was used. 10 μl of EIA buffer solution, 10 μl of COX-2 enzyme, and 10 μl of Heme were added to a 96-well plate coated with prostaglandin antibody, 10 μl of buffer solution was added to the well of the well, and 10 μl of the sample well was added so that the final concentration of the sample became 10 ppm Thereafter, reaction was carried out at 25 DEG C for 5 minutes. The colorimetric substrate and the arachidonic acid substrate were added thereto, and the absorbance was measured at a wavelength of 590 nm after 2 minutes. The degree of inhibition of COX-2 activity was calculated and compared with the calculation method (activity inhibition rate (%) = (1 - sample absorbance / control absorbance) X 100). The IC ₅₀ values are shown in Table 4 and FIG. 7 in ppm in terms of the minimum concentration required to inhibit 50% of the generated enzyme.

Sample COX-2 inhibitory activity IC ₅₀ (ppm) Burdock extract
(95% of actinogen) 992.08 98% tannic acid 1.65 AT101 1.34

COX-2 Inhibition Activity As shown in Table 4 and FIG. 7, 98% tannic acid showed 50% inhibitory activity at 1.65 ppm, and complex (AT101) showed 50% inhibitory activity at 1.34 ppm.

Experimental Example  8: Mast cell-cytotoxicity experiment MTT  assay)

SD rats at approximately 6 weeks of age, which had been subjected to a weekly purifying period, were sacrificed and used in the experiment.

30 mL of HEPES-DMEM was injected into the abdominal cavity of the sacrificed rats, and the abdominal massage was performed for 3 minutes. HEPES-DMEM in which mast cells were suspended in the peritoneal cavity was taken using a 10 mL syringe and washed three times with clean medium. The number of mast cells was measured by a microscope using a hemocyte counter, and the cells were dispensed at a rate of 2 × 10 ⁵ cells / well in a 96-well cell culture plate. The treated cells were treated at different concentrations, except for the control (0 ppm), and cultured at 37 ° C for 24 hours. After 24 hours, 20 쨉 l of a 0.2% MTT solution was added to all wells, followed by reaction at 37 째 C for 2 hours. After the reaction, all of the supernatant was removed and 200 μl of DMSO was added to all wells. The formazan produced at room temperature for 10 minutes was dissolved and the absorbance was measured at 570 nm using an ELISA reader. The cell survival rate (%) is shown in Figs. 8 and 9 using the calculation method (cell survival rate (%) = (sample treated group absorbance / control absorbance) X 100).

As shown in FIGS. 8 and 9, more than 70% of the cells survived at less than 100 ppm of 95% actinin (as a burdock extract tablet concentrate), and 98% tannic acid was 80% or less at 100 ppm or less, The above cells survived.

Experimental Example  9: Histamine glass inhibition experiment

SD rats at approximately 6 weeks of age, which had been subjected to a weekly purifying period, were sacrificed and used in the experiment.

30 mL of HEPES-DMEM was injected into the abdominal cavity of the sacrificed rats, and the abdominal massage was performed for 3 minutes. HEPES-DMEM in which mast cells were suspended in the peritoneal cavity was taken using a 10 mL syringe and washed three times with clean medium. The number of mast cells was measured by a microscope using a hemocyte counter, and the cells were dispensed into a 24-well cell culture plate at 5 × 10 ⁵ / well. Samples were treated at different concentrations except for the normal group and the control group, and then cultured at 37 ° C and 5% CO ₂ for 10 minutes. Subsequently, Compound 48/80 was treated at a concentration of 5 μg / ml in all wells except for the normal group, and cultured at 37 ° C and 5% CO ₂ for 20 minutes. The supernatant of the cell culture was quantitated and used for the experiment .

Histamine was measured using the Histamine ELISA Kit Instructions (NEOGEN com. USA). The cell culture supernatant solution per well was diluted to a final protein amount of 1 mu g in a 96 well plate, and 50 mu l of the diluted solution was dispensed. Enzyme conjugate (50 μl) was added to all wells, reacted at room temperature for 45 minutes, and washed three times with 200 μl of washing buffer. Thereafter, 150 쨉 l of K-blue substrate was dispensed into all wells, reacted at room temperature for 30 minutes, and absorbance was measured at 650 nm with an ELISA reader. The amount of histamine was quantitated using a standard curve (0, 2.5, 5, 10, 20, 50 ng / 50 μl). The glass inhibition rate (%) was compared with the calculation method (glass inhibition rate (%) = 1 - sample absorbance / control absorbance) X 100). IC ₅₀ values are shown in Tables 5 and 10 in ppm in the minimum concentration required to inhibit 50% free histamine. In Fig. 10, the numbers in parentheses indicate the sample throughput (ppm).

Sample Histamine inhibitory activity IC ₅₀ (ppm) Burdock extract
(95% of actinogen) 8.34 98% tannic acid 19.88 AT101 2.08

As shown in Table 5 and FIG. 10, 95% of the actinin (as a burdock extract tablet concentrate) had a 50% inhibitory effect at 8.34 ppm, and the complex (AT101) had a 50% inhibition at 2.08 ppm, Free inhibition.

Experimental Example  10: Raw264 .7 Inhibition of cell-inflammatory cytokine expression

Mouse macrophages (Raw 264.7 cells) were plated in 12-well cell culture plates at 1 × 10 ⁶ cells / well and then pre-cultured at 37 ° C., 5% CO ₂ for 24 hours. After 24 hours, the medium used for the pre-culture was removed, the sample was replaced with the medium supplemented with each concentration, and then cultured at 37 ° C and 5% CO ₂ for 10 minutes. / Ml and cultured at 37 ° C and 5% CO ₂ for 20 hours to induce inflammation. After 20 hours of culture, the cell culture supernatant was quantitated and used in the experiment.

Cytokines (TNF-α, IL-6) were quantitated using a Mouse ELISA Ready Set GO kit (eBioscience com., USA). Capture antibodies were diluted 250-fold in buffer and dispensed in a 96-well plate at a rate of 100 μl. The 96-well plate was coated at 4 ° C for 12 hours. After 12 hours, each well was washed three times with 200 占 퐇 of PBST, and 200 占 퐇 of buffer was added thereto and blocked at room temperature for 1 hour. After 1 hour, the buffer solution was removed and 100 占 퐇 of the culture supernatant diluted to a protein amount of 30 占 퐂 / 100 占 퐇 was dispensed, followed by reaction at room temperature for 2 hours. After 2 hours, the wells were washed three times with 200 μl of PBST, and the detection antibody was diluted 250-fold in the buffer and 100 μl each was dispensed. The reaction was then allowed to proceed at room temperature for 1 hour. After 1 hour, each well was washed with 200 쨉 l of PBST three times, HRP was diluted 250-fold in buffer, and 100 쨉 l of each was dispensed, followed by reaction at room temperature for 30 minutes. After 30 minutes, each well was washed with 200 PB of PBST three times, and 100 쨉 l of TMB solution was dispensed, followed by reaction at room temperature for 15 minutes. After 15 minutes, 50 μl of 2N H ₂ SO ₄ was added to stop the reaction, and the absorbance at 450 nm was measured using an ELISA reader. The amount of cytokine was quantitated using a standard curve (0, 3.125, 6, 25, 12.5, 25, 50, 100 pg / 100 μl).

(% Inhibition rate = 1 - sample absorbance / control absorbance) X 100). The IC ₅₀ values are shown in Table 6 and Fig. 11 (inhibitory effect on TNF-α expression) and in Table 7 and Fig. 12 (inhibitory effect on IL-6 expression), respectively, in the minimum concentration required to inhibit 50% expression. 11 and 12, the numbers in parentheses indicate sample throughput (in ppm).

Sample TNF-α expression inhibitory activity IC ₅₀ (ppm) Burdock extract
(95% of actinogen) 17.64 98% tannic acid 41.55 AT101 11.61

Sample IL-6 expression inhibitory activity IC ₅₀ (ppm) Burdock extract
(95% of actinogen) 11.01 98% tannic acid 2.39 AT101 1.66

As shown in Table 6, FIG. 11, Table 7 and FIG. 12, AT101 inhibited TNF-a by 50% at a concentration of 11.61 ppm, At a concentration of 1.66 ppm IL-6 was inhibited by 50%.

Experimental Example  11: Manual skin Anapelaxis (Passive cutaneous anaphylaxis) Animal room Humans blue pigment  Antibody-antigen reaction inhibition experiment)

ICR mice were tested for 7 weeks in each group. After a week of purifying period, 10 쨉 l of anti-DNP IgE antibody was injected subcutaneously at 1.2 쨉 g / ml in both ears of mice. After 47 hours, they were dissolved in 200 μl of PBS to the desired concentration of the sample, and orally administered in 200 μl each (using a promethazine hydrochloride as a positive control). After 1 hour, 250 μl of antigen + dye solution (DNP-albumin antigen 1 mg + Evans blue 5 mg / ml) was intravenously injected. After 30 minutes, mice were sacrificed and both ears were used in the experiment. Both ears were immersed in 1 ml of 1N KOH solution and Evans blue was eluted at 37 ° C for 12 hours. Reaction stop solution (0.6NH ₃ PO ₄ : Acetone = 5: 13), centrifuged at 3000 rpm for 15 minutes, and the absorbance of the supernatant was measured at 620 nm.

The amount of evans blue leaking from both ear veins was quantified using standard curves of standard (31.25, 62.5, 125, and 250 ppm) and shown in Figures 13 and 14.

Passive cutaneous anaphylaxis As shown in FIG. 13 and FIG. 14, in the animals, 20 mg / kg and 50 mg / kg of 95% actinin (as a concentrate of burdock extract concentrate) were 45.5 Kg and 50 mg / kg, respectively, inhibited 56.2% and 80.4%, respectively. The intake of 20 mg / kg and 50 mg / kg of compound AT101 was 39.9% and 86.1% %. 13 and 14, the numbers in parentheses indicate the sample throughput (unit: mg / kg).

Experimental Example  12: Manual skin Anapelaxis (Passive cutaneous anaphylaxis) Animal experiments Leukotriene , Prostaglandin E ₂  Concentration measurement)

ICR mice were tested at 7 weeks for each group. After a week of purifying period, 10 [mu] l of anti-DNP IgE antibody was injected subcutaneously into the both ears of mice at a concentration of 1.2 [mu] g / ml. After 47 hours, they were dissolved in 200 μl of PBS to the desired concentration of the sample and 200 μl of each solution was orally administered (using promethazine hydrochloride as a positive control). After 1 hour, 250 μl of antigen + dye solution (DNP-albumin antigen 1 mg + Evans blue 5 mg / ml) was intravenously injected. After 30 minutes, mice were sacrificed and blood was collected. The serum was centrifuged at 10000 rpm for 10 minutes, and the protein was quantified and used for the experiment.

Leukotriene and prostaglandin E ₂ were measured using an EIA kit (Enzo, USA). 50 μl of Conjugate, 50 μl of leukotriene or prostaglandin E ₂ antibody solution, and 100 μl of sample were dispensed into a 96-well plate, followed by reaction at room temperature for 2 hours. After 2 hours, all the wells were emptied, 300 μl of wash buffer was added, and washed three times. Then, 200 μl of the pNP substrate solution was dispensed into all the wells and allowed to react at room temperature for 45 minutes. After adding 50 μl of stop solution to all wells, the absorbance was measured at 405 nm using an ELISA reader. The amounts of leukotriene or prostaglandin E ₂ were quantified using the standard curves (3.9, 7.8, 15.6, 31.25, 62.5, 125, 250 pg / 100 μl) and shown in FIG. 15 and FIG. Significance was shown by t-test statistical analysis (p <0.05 compared with # = normal group, * = p <0.05 compared with control group). In Figures 15 and 16, the numbers in parentheses indicate the sample throughput (unit: mg / kg).

As shown in FIG. 15, 95% of actininin (as a concentrate of burdock extract concentrate) inhibited 43.4% and 52.5% of the intakes of 20 mg / kg and 50 mg / kg of mouse serum, respectively Kg, and 50 mg / kg, respectively, inhibited 47.2% and 65.8%, respectively, and that of the compound AT101 20 mg / kg and 50 mg / kg, respectively, inhibited 73.4% and 89.2%, respectively. As shown in FIG. 16, the amounts of Prostaglandin E _{2 in} mouse serum were 40.7% and 50.0%, respectively, in the case of 20 mg / kg and 50 mg / kg of 95% actinin (as a concentrate of burdock extract) 43.6% and 51.4% of the intake of 98% tannic acid 20 mg / kg and 50 mg / kg were inhibited respectively, and 57.3% and 74.4% of the intakes of the compound AT101 20 mg / kg and 50 mg / kg were inhibited, respectively.

Experimental Example  13: DNCB (2,4- Dinitrochlorobenzene ) Atopy animal experiment

BALB / c mice were weighed 6 weeks in each group. After a week of purifying period, the mice were sacrificed to remove dorsal hair and ear hair. Then, 1% of DNCB was added to a mixture of acetone and olive oil at a ratio of 4: 1, and 100 μl , And 20 [mu] l of the solution was applied to both ears to sensitize the cells (in the case of the normal group, only a mixture of acetone and olive oil in a ratio of 4: 1 was applied). Then, 0.2% DNCB was added to a solution prepared by mixing acetone and olive oil at a ratio of 4: 1, and 100 μl was applied to the back region three times in total every 2 days, and 20 μl was applied to both ears Group, only a solution of acetone and olive oil mixed at a ratio of 4: 1 is applied). Then, 200 μl of PBS was dissolved or dissolved in 200 μl of PBS to obtain a desired concentration of the sample for 7 days. Oral 200 μl of PBS was orally administered to each of the normal and control groups, and prometazine hydrochloride was used as a positive control. The visual evaluation score was measured once a week, and the treatment effect was measured in Fig. 17 and the edema edema was measured in Fig. 18 as a graph.

In addition, the treatment effect of measuring the spleen and the ear weight at the sacrifice is shown graphically in Figs. 19 and 20, and the leukocyte, eosinophil, and basophil count are measured at sacrifice and graphically shown in Figs. 21 to 23 . At the time of sacrifice, tissues were harvested, lysed in a lysis buffer, and pulverized. The inflammatory cytokines (TNF-α, IFN-γ and IL-17) were measured using an ELISA kit (ebioscience Com. USA) , And the results are shown in Figs. 24 to 26. Significance was shown by t-test statistical analysis (p <0.05 compared with # = normal group, * = p <0.05 compared with control group). 23, the actinin content of the rhizome extract was 71%.

As shown in FIG. 17, the results of visual examination of the DNCB atopic animal showed that atopic dermatitis induced by atopic dermatitis, which was caused by inflammation, bleeding, edema, As a result, after 3 weeks, each sample was orally administered once a day for a total of 7 days, and then treated with 50 mg / kg of 95% actininin (as a concentrated burdock extract concentrate) In the group treated with 98 mg tannic acid 50 mg / kg, the compound AT101 20 mg / kg, the compound AT101 50 mg / kg, and the positive control protein 1 mg / kg, inflammation, hemorrhage, .

DNCB Atopic Animal Experiments As shown in FIG. 18, ear thickness measurements showed that ear thickness increased due to edema over the 2nd and 3rd weeks of disease induction in all the atopy-induced groups except the normal group After 3 weeks, each sample was orally administered once a day for a total of 7 days. As a result, 95% of the actininin (as a concentrated burdock extract concentrate) was administered at a dose of 20 mg / kg, 95% (50 mg / kg), 50 mg / kg of 98% tannic acid, 20 mg / kg of Compound AT101, 50 mg / kg of Compound AT101, and 1 mg / kg of prometaazine as a positive control, ㎏ treatment group showed recovery of ear edema.

In the DNCB atopic animal test, the weight of the spleen was significantly increased in the control group (Fig. 19) and the ear weight measurement result (Fig. 20) The ear weight was significantly increased compared with the normal group. On the other hand, each sample was orally administered once a day for a total of 7 days, and then treated with 50 mg / kg of 95% actininin (as a concentrate of burdock extract concentrate) and 50 mg / kg of 98% tannic acid , The compound AT101 50 mg / kg, and the positive control protein 1 mg / kg, the immune system was stabilized and the weight of the spleen was reduced and the ear edema was recovered and the ear weight was reduced.

DNCB atopic animal test As shown in FIG. 21 to FIG. 23, leukocyte, eosinophil, and basophil counts were significantly increased in the control group due to the immune system activity. On the other hand, when each sample was orally administered for 7 days in total for 1 day, 50 mg / kg administration group of 95% actinin (as a concentrate of burdock extract concentrate), 50 mg / kg of complex AT101 group, In the positive control group, 1 mg / kg, the immune system was stabilized and the number of leukocytes, eosinophils and basophils decreased.

DNCB atopic animal test tissue Inflammatory cytokine analysis showed that the expression of inflammatory cytokines (TNF-α, IFN-γ, IL-17) was significantly decreased in the control group due to the immune system activity as shown in FIG. 24 to FIG. 26 Compared with the control group. On the other hand, when each sample was orally administered once a day for a total of 7 days, the immune system was stabilized in the 50 mg / kg administration group of Compound AT101, and the expression of inflammatory cytokines (TNF-α, IFN-γ and IL-17) All showed a decrease.

As described and demonstrated in detail above, the present invention provides a natural extract compound (AT101) comprising actininin and tannic acid as active ingredients having antiinflammation, inhibition of histamine release and alleviating allergic diseases. AT101 inhibits 5-lipoxygenase (5-LO) and cyclooxygenase (COX-2) and inhibits the expression of inflammatory cytokines (TNF-α, IL-1β and IL-6) , And inhibited the release of histamine, the most important mechanism in allergic diseases, to an excellent extent. Animal experiments have shown that by inhibiting Evans blue leakage, leucotriene generation, and prostaglandin E ₂ production, it mitigates passive skin anaphylaxis, reduces blood leukocyte counts, inhibits the inflammatory cytokine expression in tissues and alleviates atopic symptoms by DNCB, It is possible to improve the allergic reaction as well as to exhibit the antiallergic ability similar to that of the positive control, i.e., prometadine, so that the composition can act as a composition capable of effectively improving allergic diseases.

While the invention has been shown and described with reference to certain exemplary embodiments thereof, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined by the appended claims.

N: normal group C: control group
A1: burdock extract (actigenein 0.315%) A2: burdock extract (actigenein 43.24%)
A3: Burdock extract (actinine 95.00%)
T1: 40% Tannic acid T2: 70% Tannic acid
T3: 98% Tannic acid
AT1: 95% actinin (as a burdock extract tablet concentrate): 98% tannic acid = 2: 1
AT2: 95% actinin (as a burdock extract tablet concentrate): 98% tannic acid = 4: 1
AT3: 95% actinin (as a burdock extract tablet concentrate): 98% tannic acid = 5: 1
AT4: 95% actinin (as a burdock extract tablet concentrate): 98% tannic acid = 8: 1
P: prometazine

Claims (9)

A composition for alleviating allergic diseases, which comprises actinin and tannic acid as active ingredients. The method according to claim 1,
Wherein the actininase is obtained from an extract of burdock, allium, mackerel duck, forsythia, whitehead, casualty, rockfall or the like. The method according to claim 1,
Wherein the actininase is 95% actinin (as a concentrate of burdock extract). The method of claim 3,
A composition for alleviating allergic diseases, comprising 95% actinin (as a concentrated burdock extract concentrate): 98% tannic acid in a weight ratio of 2 to 10: 1. A health functional food for alleviating allergic diseases, which comprises a composition according to any one of claims 1 to 4. 6. The method of claim 5,
A health functional food for alleviating allergic diseases, which comprises the composition according to any one of claims 1 to 4 in an amount of 0.1 to 100% by weight. The method according to claim 6,
A health functional food for relieving allergies such as tablets, capsules, pills or liquids. A coating agent for alleviating allergic diseases, which comprises a composition according to any one of claims 1 to 4. The coating agent for alleviating allergic diseases according to claim 8, which comprises 0.1 to 100% by weight of the composition according to claim 1 or 2.

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