KR102035321B1 - Composition for alleviating allergic disease - Google Patents

Abstract

The present invention relates to a composition for allergic disease alleviation comprising actinizenin and tannic acid as an active ingredient, and excellent in anti-inflammatory effect and histamine release, having alleviating allergic diseases such as rhinitis, asthma and atopic dermatitis. , Characterized in that it comprises actinigen and tannic acid as an active ingredient.

Description

Composition for allergy disease alleviation {Composition for alleviating allergic disease}

The present invention relates to a composition for allergic disease alleviation, in particular, it contains actinogen and tannic acid (tannic acid) as an active ingredient, and excellent in anti-inflammatory effect and histamine release, alleviating allergic diseases such as rhinitis, asthma, atopy It relates to a composition for allergy disease alleviation.

Allergic symptoms are when allergens from outside are introduced into the body, and IgE antibodies to antigens are produced in immune cells, and after IgE binds to IgE receptors on mast cells and platelets, When the antigen introduced into IgE binds to mast cells or platelets, it is known that various substances such as histamine and serotonin are released freely.

Typical examples of allergic symptoms include skin-related abnormalities that can be referred to collectively as a series of atopic diseases, such as dry skin, keratinization, itching, and inflammation caused by secondary microbial infections. This will happen. Allergic symptoms, mainly in the elderly, children, and infants, are characterized by chronic diseases caused by repeated exposure to external environmental factors. The most significant cause of skin-related abnormalities is due to various allergens caused by urbanization and industrialization of living environment, and recently, it occurs in various forms regardless of seasonal characteristics.

Therefore, various types of drugs and preparations have been developed according to therapeutic purposes such as moisturizers, antibacterial agents, anti-inflammatory agents, and steroid hormones, but long-term repeated use is inevitable.

The type of preparation mainly used for the management of skin-related abnormalities is an emollient that forms a thin oil film on the skin to maintain moisture and flexibility of the skin, external ointment with hydrocortisone, antihistamine oral administration, steroid or antihistamine Bandages containing, oil-based external preparations for the purpose of improving dryness, diet and the like have been utilized.

Korean Laid-Open Patent Publication No. 1992-0021158 (name of the invention: Allergic disease prevention and treatment composition containing an extract of lettuce) discloses an allergic disease prevention and treatment composition containing an extract of lettuce.

Republic of Korea Patent Registration No. 10-0821926 (name of the invention: a tick allergen neutralizing agent comprising a plant extract and a composition comprising the same) "The present invention relates to a tick allergen neutralizing agent comprising a plant extract and a composition comprising the same As regards the present invention, natural plant extracts and plant essential oils, which can neutralize allergens produced by major dust mite species worldwide and cause various diseases, and one or two or more of the active substances contained therein, or It provides a protein neutralizing agent composition containing an essential oil and a method for producing the same.

Korean Laid-Open Patent Publication No. 10-2010-0087785 (name of the invention: skin disease inhibiting composition using a duct and its preparation method) "The present invention relates to a duct extract useful for treating skin diseases, more specifically The present invention relates to duct extract and its composition useful for the prevention and treatment of skin diseases such as atopy and psoriasis, by confirming that duct extract is excellent in antioxidant effect and epidermal growth inhibitory effect. Since the skin-related diseases and detoxification are known to be excellent, the present invention is an excellent invention useful for preventing and treating skin diseases such as atopy and psoriasis. ”

Republic of Korea Patent Publication No. 10-0566542 (name of the invention: health food composition for the prevention and treatment of allergic rhinitis and cold and a method of manufacturing the same) "The present invention is roots, dermis, licorice, Schisandra chinensis, Euiin, Eoseongcho , Astragalus, Macmundong, three hundred seconds, gyeji, golden, peppermint and baekryeum as an active ingredient for allergic rhinitis and colds, and a health food composition for the prevention and treatment and a method of manufacturing the same. "

However, there is still an urgent 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 an anti-inflammatory effect for the fundamental improvement of allergic diseases suffered by modern people, and after diligently researching to develop a composition that maximizes the alleviation effect of allergens by selecting natural materials that help inhibit histamine release, acti Including xenin and tannic acid as active ingredients, and excellent in anti-inflammatory effect and histamine free inhibition, we developed a composition for allergic diseases alleviating allergic diseases such as rhinitis, asthma, atopic dermatitis, the effect of this composition in vitro The present invention has been completed by proving through in vitro and in vivo experiments.

The composition for allergy disease alleviation according to the present invention includes actinigen and tannic acid as active ingredients.

According to the present invention, it contains actinizenin and tannic acid (tannic acid) as an active ingredient, and excellent in anti-inflammatory effect and histamine free inhibition, to provide an alleviating effect of allergic diseases such as rhinitis, asthma, atopy.

1 is a graph showing a comparison of histamine free inhibition of natural product extracts.
Figure 2 is a graph showing a comparison of histamine free inhibition of burdock extracts.
3 is a graph showing a comparison of COX-2 inhibitory activity according to tannic acid concentration.
FIG. 4 is a graph showing the comparison of histamine free inhibition according to 95% actinizenin (as burdock extract purified concentrate) and 98% tannic acid combination ratio.
5 is a graph showing the 5-lipoxygenase inhibitory effect of AT101 according to the present invention.
Figure 6 is a graph showing the inhibitory effect of the induced NO production enzyme (iNOS) of AT101 according to the present invention.
7 is a graph showing the COX-2 inhibitory effect of AT101 according to the present invention.
FIG. 8 is a graph showing the results of 95% actinigenin (as burdock extract purified concentrate) MTT assay.
9 is a graph showing the results of 98% tannic acid MTT analysis.
10 is a graph showing histamine free inhibitory efficacy.
11 is a graph showing the effect of TNF-α expression inhibition.
12 is a graph showing the IL-6 expression inhibitory effect.
13 is a graph showing the Evans blue leakage inhibitory effect.
14 is a photograph of Evans blue leak suppression.
15 is a graph showing the leukotriene inhibitory effect in serum.
16 is a graph showing the prostaglandin E ₂ inhibitory effect in serum.
17 is a graph showing the visual score of DNCB animal experiments.
18 is a graph showing ear thickness change results in DNCB animal experimental ear edema.
19 is a graph showing the results of changes in spleen weight of DNCB animals.
20 is a graph showing the results of DNCB animal experiment ear weight change.
21 is a graph showing the results of changes in the white blood cell count during DNCB animal experiment blood analysis.
22 is a graph showing the results of changes in eosinophil count during blood analysis of DNCB animals.
Figure 23 is a graph showing the change in basophil number in the blood analysis of DNCB animal experiments.
Figure 24 is a graph showing the results of TNF-α change in the DNCB animal experiments ELISA tissue analysis.
25 is a graph showing the results of IFN-γ change in DNCB animal test ELISA tissue analysis.
Figure 26 is a graph showing the results of IL-17 change in the DNCB animal experiment ELISA tissue analysis.

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

The allergic disease relief composition according to the present invention is characterized in that it comprises actinigen and tannic acid as an active ingredient. Hereinafter, the composition according to the present invention containing actinigen and tannic acid as an active ingredient is abbreviated as AT101.

Symbols in the figures are in accordance with the legend described in the following [Description of Symbols] unless otherwise specified, and the numbers in parentheses written in the symbols refer to the addition amount (in ppm), unless otherwise specified. And in the case of Fig. 13 to 26, it means the dosage (unit mg / kg).

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

Actigenin can be obtained from extracts of various plants, in particular, extracts such as, for example, burdock, pontoon, macmundong, forsythia, baekduong, casualty, rockfall, etc. Hereinafter will be described based on the example of the burdock extract.

The burdock extract is washed with the burdock raw material, dried in a shade, crushed or pulverized, and then 20 to 100 ° C. with water, ethanol or a mixed solvent thereof, preferably water, 2 to 20 times the original weight. Extraction method such as hydrothermal extraction, ultrasonic extraction, reflux cooling extraction, etc. repeatedly at 1 to 10 times, preferably 2 to 5 times, for 1 hour to 2 days, preferably 2 to 8 hours at an extraction temperature of 50 to 95 ℃ Preferably by carrying out hydrothermal extraction.

Burdock ( Arcium) lappa L. ) is a plant, genus plant, dicotyledon, campanula, asteraceae, naturalized in Europe. The varieties include long roots, thick coarse farmland, good quality flesh, and short roots. The root contains inulin and some palmitic acid. In Europe, it is used as a diuretic and antiperspirant, and the seed is used as a diuretic when there is swelling, and as an antidote for sore throats and poisons. It is grown in Japan and wild in Europe, Siberia, and northeastern China. In oriental medicine, seeds are used as medicinal herbs. The drug is effective in sweeping wind fever and preemptive pneumonia, dissipation, and detoxification. Wind heat seawater, sore throat, rubella, etc. Used. The obtained burdock extract may be filtered, concentrated under reduced pressure or concentrated in vacuo to obtain an extract concentrate, and additionally degreased using n-hexane and effective using a Diaion HP-20 ion resin and a Sephadex LH-20 column. It is possible to increase the extraction yield of the component actinigen.

In the case of tannic acid, an anti-inflammatory effect has already been demonstrated in many literatures, and purified extracts of more than 95% content can be easily purchased and used.

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

Specifically, the composition for allergic disease alleviation according to the present invention is 95% actinigenin (as burdock extract tablet concentrate): 98% tannic acid in a weight ratio of 2 to 10: 1, preferably 4 to 8: 1, more preferably Is characterized by including 5: 1.

In addition, the method for producing a composition for alleviating allergic diseases according to the present invention comprises the steps of: (1) obtaining a burdock extract from a burdock raw material; And (2) 95% actinizenin (as burdock extract purified concentrate): actinizenin and tannic acid to 98% tannic acid in a weight ratio of 2 to 10: 1, preferably 4 to 6: 1, more preferably 5: 1 Mixing with; characterized in that it comprises a.

5-lipoxygenase (5-lipoxygenase) inhibitory effect of the composition obtained by the same method, inducible Nitric oxide synthase (iNOS) inhibitory effect, COX-2 (cyclooxygenase-2) 2)) inhibitory effect, histamine free inhibitory effect and inflammatory cytokine (TNF-α, IL-1β, IL-6) expression inhibitory effect was measured. In addition, animal experiments were conducted to investigate the alleviation effects of allergic diseases.

The composition for allergy disease alleviation according to the present invention may be commercialized in various forms. Examples of commercialization include, for example, allergic disease alleviation health functional food containing the composition as an active ingredient, allergic disease alleviation coating agent, allergic disease alleviation general food, etc., these are 0.1 to 100% by weight It may be included, but it is to be understood that the invention is not intended to be limited thereto. These may further comprise suitable carriers, excipients and diluents commonly used in the preparation of natural product compositions.

In addition, the composition for allergy disease alleviation according to the present invention can be formulated in various forms. Examples of formulations include, for example, tablets, capsules, pills, solutions, and the like, but it should be understood that the present invention is not intended to be limited thereto.

The compositions according to the invention can each be formulated and used in oral formulations of powders, granules, tablets, capsules, suspensions, emulsions, syrups and the like according to conventional methods. Carriers, excipients and diluents that may be included in the compositions comprising the compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate , Calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, honey, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid preparations may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, or the like. Mix is prepared. In addition to simple excipients, lubricants such as magnesium stearate are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. .

Preferred dosages of the compositions of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the composition of the present invention is preferably administered in 0.1 to 1g per day. Administration may be once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

Since the composition of the present invention does not have toxicity and side effects, it can be used with confidence even for long-term use for prophylactic purposes.

Hereinafter, the present invention will be described in detail by Examples and Experimental Examples.

However, the following Examples and Experimental Examples are only for illustrating the present invention, and the content of the present invention is not limited to the following Examples and Experimental Examples.

Test Example  1: Selection of Natural Products and Preparation of Extracts

20 kinds of natural products that are expected to have anti-allergic effects through literature research (Cinnamon, Falconia, Hyeonggae, Gilgyeong, Forsythia, Self-designation, Seonmo, Burdock, Macmun-dong, Gardenia, Baekduong, Casualties, Cypress, Chilgapdam, Darae, Yugeunpi, Rockfall, guava, white paper, evening primrose, aloe) were selected and purchased from a raw material supply company. In order to prepare a natural extract, each natural product 1kg each dried, and then crushed 100g of the powder obtained in a household bottle, and then put 1000ml of 80% edible alcohol and extracted twice at 80 ℃ for 4 hours.

Experimental Example  1: Comparison of Histamine Free Inhibitory Effects

Approximately 6 weeks old SD rats, which had undergone one week of purification, were sacrificed and used in the experiment.

30 ml of HEPES-Dulbecco's modied Eagle's medium (HEPES-DMEM) was injected into the abdominal cavity of the sacrificed rats, followed by an abdominal massage for 3 minutes. Again, HEPES-DMEM with mast cells suspended in the abdominal cavity using a 10 ml syringe was washed three times with clean medium. After measuring the number of mast cells under a microscope using a hemacytometer, 5 × 10 ⁵ / well was dispensed into a 24-well cell culture plate. The samples were treated with 10ppm except the normal group and the control group, and then cultured at 37 ° C. and 5% CO ₂ for 10 minutes. Thereafter, all wells except the normal group were treated with Compound 48/80 at a concentration of 5 µg / ml, and then cultured at 37 ° C. and 5% CO ₂ for 20 minutes, and the cell culture supernatant was used for protein quantification.

Histamine was measured using Histamine ELISA Kit Instructions (NEOGEN com, USA). Cell culture supernatants per well were diluted in a 96 well plate to a final protein amount of 1 μg and 50 μl were dispensed. 50 μl each of the Enzyme conjugate was dispensed into all wells and allowed to react at room temperature for 45 minutes, followed by washing three times with 200 μl of washing buffer. Thereafter, 150 μl of K-blue substrate was dispensed into all wells, and then allowed to react at room temperature for 30 minutes, followed by measurement of absorbance at 650 nm with an ELISA reader. The amount of histamine was quantified using a standard (0, 2.5, 5, 10, 20, 50 ng / 50 μl) standard curve and the results are shown in FIG. 1.

As a result of the histamine free inhibition experiment, as shown in FIG. Was the best.

Experimental Example  2: HPLC  analysis - Actigenin  content

HPLC analysis was performed to investigate the contents of actinigenin of 8 natural extracts (Yeongyo, Forsythia, Burdock, Macmun-dong, Baekduong, Casualties, Chilgapdam, Rockfall, etc.) with excellent histamine free inhibitory effect. RP-HPLC used Model code 6CE (Waters, Milford, MA). Pump is Model code 60F, detector is Waters 2414, auto sampler is Waters 717 _plus , degassing unit is Inline Degasser-AF, column is Phenomenex's Luna C18 Column (250 × 4.6 mm, particle size 5㎛) ) Was used. Actigenin was analyzed for methanol and tertiary distilled water at 55:45 (v / v), filtered with a 0.45 μm Millipore Durapore filter and then degassed by sonication prior to mobile phase use. RP-HPLC elution was an isocratic elution method using 1.0 ml / min flow rate, 10 μl sample injection rate, and column temperature at 30 ° C. for 35 minutes at 280 nm. 0.2 μm) was used for filtering, and the results are shown in Table 1 (HPLC analysis—actinigen content).

Natural product extract Actigenin content (%) Fellowship 0.284 forsythia 0.262 burdock 0.315 Macmundong 0.226 Baekduong 0.194 Casualties 0.246 Chirping Wall 0.252 Rockfall 0.217

As a result of HPLC analysis, as shown in Table 1, all 8 kinds of natural products (Yeongyo, Forsythia, Burdock, Macmundong, Baekduong, Casualties, Chilgapdam, Rockfall, etc.) all contained actinogen as an active ingredient. The highest level of actinigen content of burdock extract was 0.315%.

Example  1: Purification of Burdock Extract- Actigenin

In order to increase the actinigen content of burdock, the active ingredient of the extract, it was degreased by adding the same amount of n-hexane, concentrated to obtain 20g of powder, dissolved in 1L of 10% MeOH, pectinase and cellulase Each of 0.3 g of cellulase was added and hydrolyzed with stirring at 50 ° C. for 12 hours. The hydrolyzed extract was passed through a Diaion HP-20 ion resin column to obtain a portion eluted with 80% MeOH solvent, and then passed through a Sephadex LH-20 column to select the fraction containing the active ingredient and 40 ° C. with a vacuum rotary concentrator. Concentrated under reduced pressure at and then dried to obtain Burdock extract (Actininin 43.24%)

Purified burdock extract (95% actinigen) was also purchased.

Experimental Example 3: 95% Of actigenin (burdock extract purified concentrate)  Histamine Free Inhibitory Effects

Approximately 6 weeks old SD rats, which had undergone one week of purification, were sacrificed and used in the experiment.

30 ml of HEPES-DMEM was injected into the abdominal cavity of the sacrificed rats, followed by an abdominal massage for 3 minutes. Another 10 ml syringe was used to take HEPES-DMEM suspended in mast cells in the abdominal cavity, and then washed three times with clean medium. After measuring the number of mast cells under a microscope using a hemocytometer, 5 x 10 ⁵ / well was dispensed in a 24-well cell culture plate. The samples were treated with 10ppm except the normal group and the control group, and then incubated at 37 ° C. and 5% CO ₂ for 10 minutes. Thereafter, all wells except the normal group were treated with Compound 48/80 at a concentration of 5 µg / ml, and then cultured at 37 ° C. and 5% CO ₂ for 20 minutes, and the cell culture supernatant was used for protein quantification.

Histamine was measured using Histamine ELISA Kit Instructions (NEOGEN com, USA). Cell culture supernatants per well were diluted in a 96 well plate so that the final protein amount was 1 μg, and 50 μl were dispensed. 50 μl of Enzyme conjugate was dispensed into all wells and allowed to react at room temperature for 45 minutes, followed by washing three times with 200 μl of wash buffer. Thereafter, 150 μl of K-blue substrate was dispensed into all wells, and then reacted at room temperature for 30 minutes, and then absorbance was measured at 650 nm with an ELISA reader. The amount of histamine was quantified using a standard (0, 2.5, 5, 10, 20, 50 ng / 50 μl) standard curve and the results are shown in FIG. 2 (the figures in parentheses in FIG. 2 are actinogen content).

As a result of the histamine free inhibition experiment, as shown in FIG. 2, the higher the actinigen content of the burdock extract, the better the histamine free inhibition effect.

Experimental Example  4: Tannic acid  Comparison of Anti-inflammatory Effects According to Concentration-Measurement of COX-2 Inhibitory Activity

Purified tannic acid was purchased at three concentrations (40%, 70%, 98%) and the COX-2 inhibitory activity comparison experiment was conducted. COX Colorimetric Inhibitor Screening Assay Kit (Creative Biomart Com, USA) was used. 150 μl of EIA buffer, 10 μl of COX-2 enzyme, and 10 μl of Heme were added to a 96 well plate coated with prostaglandin antibody, and 10 μl of the control well was added to 10 μl of the buffer solution and 10 μm of the sample well was added to the final concentration of the sample. After that, the reaction was carried out at 25 ° C. for 5 minutes. A colorimetric substrate and arachidonic acid as a substrate were added thereto, and the absorbance was measured at a wavelength of 590 nm after 2 minutes. The degree of activity inhibition of COX-2 was compared and analyzed by the calculation method (activity inhibition rate (%) = (1-sample absorbance / control absorbance) X 100). IC ₅₀ values are shown in FIG. 3 in ppm units for the minimum concentration required to inhibit 50% of the enzymes that occurred.

As a result of the COX-2 inhibitory activity test, as shown in FIG. 3, the higher the content of tannic acid, the better the COX-2 inhibitory activity effect.

Example  2: 95% to maximize the histamine free inhibitory effect Actigenin (burdock extract refine As a concentrate ) And 98% Tannic acid  Compounding cost investigation

Approximately 6 weeks old SD rats, which had undergone one week of purification, were sacrificed and used in the experiment.

30 ml of HEPES-DMEM was injected into the abdominal cavity of the sacrificed rats, followed by an abdominal massage for 3 minutes. Another 10 ml syringe was used to take HEPES-DMEM suspended in mast cells in the abdominal cavity, and then washed three times with clean medium. After measuring the number of mast cells under a microscope using a hemocytometer, 5 x 10 ⁵ / well was dispensed in a 24-well cell culture plate. The samples were treated with 10ppm except the normal group and the control group, and then incubated at 37 ° C. and 5% CO ₂ for 10 minutes. Thereafter, all wells except the normal group were treated with Compound 48/80 at a concentration of 5 μg / ml and incubated at 37 ° C. and 5% CO ₂ for 20 minutes, and the cell culture supernatant was used for quantifying proteins. .

Histamine was measured using Histamine ELISA Kit Instructions (NEOGEN com, USA). 50 µl aliquots of the cell culture supernatant per well were diluted to 1 µg in 96-well plates. 50 μl of Enzyme conjugate was aliquoted into all wells and allowed to react at room temperature for 45 minutes, followed by washing three times with 200 μl of wash buffer. Thereafter, 150 μl of K-blue substrate was dispensed into all wells, followed by reaction at room temperature for 30 minutes, and then the absorbance was measured at 650 nm using an ELISA reader. The amount of histamine was quantified using a standard (0, 2.5, 5, 10, 20, 50 ng / 50 μl) standard curve and the results are shown in FIG. 4.

As a result of the histamine free inhibition experiment, as shown in FIG. 4, the ratio of actinogen and 98% tannic acid was 95% actinigen (as burdock extract tablet concentrate): 98% tannic acid = 5: 1 It is judged that it is advantageous in terms of functionality.

Experimental Example 5: 5 - Lipoxygenase  Inhibitory effect measurement

Lipoxygenase Inhibitor Screening Assay Kit (Cayman Chemical Com, USA) was used. Into 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 for each concentration. The reaction was performed at room temperature for 10 minutes, followed by 100 μl of chromogen. After the addition, the reaction was again performed at room temperature for 5 minutes, and the absorbance was measured at 490 nm with an ELISA reader. The degree of activity inhibition of 5-lipoxygenase was calculated using the calculation method (activity inhibition rate (%) = (1-absorbance of sample addition group / absorbance of sample no addition group) X 100). IC ₅₀ values are shown in Table 2 and FIG. 5, in ppm, for the minimum concentration required to inhibit 50% of the enzyme.

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

As a result of 5-lipoxygenase inhibitory activity test, as shown in Table 2 and FIG. 5, 98% tannic acid showed 50% inhibitory activity at 0.81 ppm, and complex 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 units / ml) enzyme and 25 µl of buffer were added to a 96 well plate, and 5 µl of the sample was added to each concentration and reacted at 25 ° C. for 15 minutes. After the reaction was completed, 10 μl of arachidonic acid substrate and 0.5 μl of Glucose dehydrogenase (GDH) were added, followed by reaction at 37 ° C. for 60 minutes, followed by dispensing 200 μl of chromogen, and then again at 37 ° C. for 60 minutes. After the reaction, the absorbance was measured at 540 nm with an ELISA reader. The degree of NOS activity inhibition was calculated using the calculation method (activity inhibition rate (%) = (1-absorbance of sample added / absorbance of sample-free group) X 100). IC ₅₀ values are shown in Table 3 and FIG. 6, in ppm, for the minimum concentration required to inhibit 50% of the enzymes generated.

Sample iNOS inhibitory activity IC ₅₀ (ppm) Burdock Extract
(95% Actigenin) 1083.59 98% tannic acid 4.61 AT101 4.21

As a result of the iNOS inhibitory activity test, as shown in Table 3 and FIG. 6, 98% tannic acid showed 50% inhibitory activity at 4.61ppm, and the complex AT101 showed 50% inhibitory activity at 4.21ppm.

Experimental Example  7: Measurement of COX-2 Inhibitory Effect

COX Colorimetric Inhibitor Screening Assay Kit (Creative Biomart Com, USA) was used. In a 96 well plate coated with prostaglandin antibody, 150 μl of EIA buffer, 10 μl of COX-2 enzyme, and 10 μl of Heme were added. The control well was added with 10 μl of buffer and the sample well was added with 10 μl so that the final concentration of the sample was 10 ppm. After that, the reaction was carried out at 25 ° C. for 5 minutes. A colorimetric substrate and an arachidonic acid substrate were added thereto, and the absorbance was measured at a wavelength of 590 nm after 2 minutes. The degree of activity inhibition of COX-2 was compared and analyzed by the calculation method (activity inhibition rate (%) = (1-sample absorbance / control absorbance) X 100). IC ₅₀ values are shown in Table 4 and FIG. 7, in ppm, for the minimum concentration required to inhibit 50% of the enzyme.

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

As a result of the COX-2 inhibitory activity test, as shown in Table 4 and FIG. 7, 98% tannic acid showed 50% inhibitory activity at 1.65 ppm, and the complex (AT101) showed 50% inhibitory activity at 1.34 ppm.

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

Approximately 6 weeks old SD rats, which had undergone one week of purification, were sacrificed and used in the experiment.

30 ml of HEPES-DMEM was injected into the abdominal cavity of the sacrificed rats, followed by an abdominal massage for 3 minutes. Another 10 ml syringe was used to take HEPES-DMEM suspended in mast cells in the abdominal cavity, and then washed three times with clean medium. After measuring the number of mast cells under a microscope using a hemocytometer, 2 x 10 ⁵ / well was dispensed in 96-well cell culture plate. Samples were treated by concentration except the control group (0 ppm) in the divided cells, and then cultured at 37 ° C. for 24 hours. After 24 hours, 20 µl of 0.2% MTT solution was added to all the wells and reacted at 37 ° C for 2 hours. After the reaction, all supernatants were removed, DMSO was added to all wells at 200 μl to dissolve formazan produced at room temperature for 10 minutes, and the absorbance was measured at 570 nm using an ELISA reader. The cell viability (%) is shown in FIG. 8 and FIG. 9 using the calculation method (% cell survival rate = (sample treated group absorbance / control absorbance) X 100).

As a result of mast cell cytotoxicity experiments, as shown in FIGS. 8 and 9, 95% actinigenin (as a burdock extract purified concentrate) survived 70% or more cells at 100 ppm or less, and 98% tannic acid was 80% or less at 100 ppm or less. The above cells survived.

Experimental Example  9: histamine free inhibition experiment

Approximately 6 weeks old SD rats, which had undergone one week of purification, were sacrificed and used in the experiment.

30 ml of HEPES-DMEM was injected into the abdominal cavity of the sacrificed rats, followed by an abdominal massage for 3 minutes. Another 10 ml syringe was used to take HEPES-DMEM suspended in mast cells in the abdominal cavity, and then washed three times with clean medium. After measuring the number of mast cells under a microscope using a hemocytometer, 5 x 10 ⁵ / well was dispensed in a 24-well cell culture plate. Samples were treated by concentration except the normal group and the control group to the divided cells, and then incubated at 37 ° C. and 5% CO ₂ for 10 minutes. Thereafter, all wells except the normal group were treated with Compound 48/80 at a concentration of 5 µg / ml and incubated at 37 ° C. and 5% CO ₂ for 20 minutes, and the cell culture supernatant was used for quantifying proteins. .

Histamine was measured using Histamine ELISA Kit Instructions (NEOGEN com. USA). 50 µl aliquots of the cell culture supernatant per well were diluted to 1 µg in 96 well plates. 50 μl of Enzyme conjugate was dispensed into all wells and allowed to react at room temperature for 45 minutes, followed by washing three times with 200 μl of wash buffer. Thereafter, 150 μl of K-blue substrate was dispensed into all wells, and then reacted at room temperature for 30 minutes, and then absorbance was measured at 650 nm with an ELISA reader. The amount of histamine was quantified using standard (0, 2.5, 5, 10, 20, 50 ng / 50 μl) standard curves. The glass inhibition rate (%) was compared and analyzed by the calculation method (glass inhibition rate (%) = 1-sample absorbance / control absorbance) X 100). IC ₅₀ values are shown in Table 5 and FIG. 10, in ppm, for the minimum concentration required to inhibit 50% glass release of histamine. The numbers in parentheses in FIG. 10 represent sample throughput in ppm.

Sample Histamine free inhibitor activity IC ₅₀ (ppm) Burdock Extract
(95% Actigenin) 8.34 98% tannic acid 19.88 AT101 2.08

As a result of the histamine free inhibition experiment, as shown in Table 5 and FIG. 10, 95% actinigenin (as a burdock extract purified concentrate) had a 50% free inhibitory effect at 8.34 ppm, and the complex (AT101) at 50% at 2.08 ppm The result was glass suppression.

Experimental Example  10: Raw264 .7 Cell-Inflammatory Cytokine Expression Inhibition Experiments

Mouse macrophages (Raw264.7 cells) were dispensed into 12 well cell cultures at 1 × 10 ⁶ cells / well and then pre-incubated for 24 hours at 37 ° C., 5% CO ₂ . After 24 hours, the medium used for pre-culture was removed and the sample was replaced with medium added at each concentration, followed by incubation for 10 minutes at 37 ° C., 5% CO ₂ , followed by 100ng of LPS except for the normal group. Inflammation was induced by treatment at a / ml concentration and incubated at 37 ° C., 5% CO ₂ for 20 hours. After 20 hours of incubation, the cell culture supernatant was quantified and used for the experiment.

Cytokines (TNF-α, IL-6) were quantified using a Mouse ELISA Ready Set GO kit (eBioscience com. USA). Capturing antibody was diluted 250-fold in buffer and dispensed into the wells of 100 μl in 96-well plates, and then 96-well plates were coated at 4 ° C. for 12 hours. After 12 hours, each well was washed three times with 200 μl of PBST, and then 200 μl of buffer solution was blocked for 1 hour at room temperature. After 1 hour, the buffer solution was removed, and 100 µl of the culture supernatant diluted so that the protein amount was 30 µg / 100 µl, and then reacted at room temperature for 2 hours. After 2 hours, each well was washed three times with 200 µl of PBST, and then 100 µl of the detection antibody was diluted 250-fold in a buffer, followed by reaction at room temperature for 1 hour. After 1 hour, each well was washed three times with 200 µl of PBST, and 100 µl of HRP was diluted 250-fold in buffer, followed by reaction at room temperature for 30 minutes. After 30 minutes, each well was washed three times with 200 µl of PBST, and then 100 µl of TMB solution was reacted at room temperature for 15 minutes. After 15 minutes, 50 μl of 2N H ₂ SO ₄ was dispensed to stop the reaction, and then the absorbance was measured at 450 nm using an ELISA reader. The amount of cytokines was quantified using standard (0, 3.125, 6,25, 12.5, 25, 50, 100 pg / 100 μl) standard curves.

Expression inhibition rate (%) was analyzed by the calculation method (expression inhibition rate (%) = 1-sample absorbance / control absorbance) X 100). IC ₅₀ values are shown in Table 6 and FIG. 11 (TNF-α expression inhibitory effect) and Table 7 and FIG. 12 (IL-6 expression inhibitory effect), respectively, in ppm units of the minimum concentration required for 50% inhibition. In Figures 11 and 12 the numbers in parentheses indicate sample throughput in ppm.

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

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

As a result of measuring the cytokine (TNF-α, IL-6) expression inhibitory results, as shown in Table 6, 11, 7 and 12, AT101 inhibited 50% of TNF-α at 11.61ppm concentration, 50% inhibition of IL-6 at 1.66 ppm concentration.

Experimental Example  11: manual skin Anacphylaxis (Passive cutaneous anaphylaxis) Animal room Evans blue pigment  Antibody-antigen reaction inhibition experiment using

Seven weeks of ICR mice were tested in each group. After a week of acclimation, 10 μl of anti-DNP IgE antibody was injected subcutaneously at 1.2 μg / ml in both ears of the mouse. After 47 hours, the solution was prepared by dissolving in 200 μl PBS to the desired concentration of the sample and administering 200 μl orally (using promethazine hydrochloride as a positive control). After 1 hour, 250 µl of the antigen + pigment solution (1 mg of DNP-albumin antigen + 5 mg / ml of Evans blue) was injected into the vein. After 30 minutes, mice were sacrificed to use both ears for the experiment. Both ears were immersed in 1 ml of 1N KOH solution to elute Evans blue at 37 ° C. for 12 hours. Reaction Stop Solution (0.6NH ₃ PO ₄ : 4 ml of acetone = 5: 13) was added and centrifuged at 3000 rpm for 15 minutes, and then the supernatant was measured for absorbance at 620 nm.

The amount of Evans blue leaking from both ear vessels was quantified using standard curves of standards (31.25, 62.5, 125, 250 ppm) and are shown in FIGS. 13 and 14.

Passive cutaneous anaphylaxis animal experiments, as shown in Figure 13 and 14, 95% actinigenin (as burdock extract tablet concentrate) 20 mg / kg and 50 mg / kg intake groups were 45.5 % And 63.5% were inhibited, and 98% tannic acid 20 mg / kg and 50 mg / kg intake groups were inhibited by 56.2% and 80.4%, respectively, and the complex AT101 20 mg / kg and 50 mg / kg intake groups were 39.9% and 86.1 respectively. % Inhibition was obtained. 13 and 14, numbers in parentheses indicate sample throughput (unit mg / kg).

Experimental Example  12: manual skin Anacphylaxis (Passive cutaneous anaphylaxis) Animal experiment (in serum Leukotrien , Prostaglandin E ₂  Concentration measurement)

Six weeks old ICR mice were tested in each group. After a week of acclimation, 10 μl of anti-DNP IgE antibody was injected subcutaneously at a concentration of 1.2 μg / ml in both ears of the mouse. After 47 hours, the solution was prepared by dissolving in 200 μl PBS to the desired concentration of the sample and administering 200 μl orally (using promethazine hydrochloride as the positive control). After 1 hour, 250 μl of the antigen + pigment solution (DNP-albumin antigen 1 mg + Evans blue 5 mg / ml) was injected into the vein. After 30 minutes, mice were sacrificed to collect blood, and serum was centrifuged at 10000 rpm for 10 minutes.

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

As a result of quantifying the amount of leukotriene in mouse serum, as shown in FIG. 15, 203.4 / kg and 50 mg / kg of 95% actinigenin (as burdock extract purified concentrate) were inhibited by 43.4% and 52.5%, respectively. , 98% tannic acid 20 mg / kg and 50 mg / kg ingested group was inhibited by 47.2%, 65.8%, respectively, complex AT101 20 mg / kg and 50 mg / kg ingested group was inhibited by 73.4%, 89.2%, respectively. As a result of quantification of the amount of Prostaglandin E _{2 in the} mouse serum, as shown in FIG. 16, the 20 mg / kg and 50 mg / kg intake groups of 95% actinigenin (as the burdock extract purified concentrate) were 40.7% and 50.0%, respectively. 43.6% and 51.4% of the 98% tannic acid 20mg / kg and 50mg / kg intake groups were inhibited, and the complex AT101 20mg / kg and 50mg / kg intake groups were inhibited by 57.3% and 74.4%, respectively.

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

Six weeks old BALB / c mice were tested in each group. After a week of purifying, hair is removed from the back and ears of the mouse, and then 100% of DNCB is added to the dorsal area three times every two days by adding 1% DNCB to a solution of acetone and olive oil in a 4: 1 ratio. , 20 μl each was applied to both ears for sensitization (in the case of the normal group, only a solution containing acetone and olive oil in a 4: 1 ratio) was included. Thereafter, 0.2% of DNCB was added to a solution of acetone and olive oil in a 4: 1 ratio, and then challenged by applying 100 μl to the dorsal region and 20 μl on both ears once every two days. In the case of the group also includes only a solution of acetone and olive oil in a 4: 1 ratio). Then, it was prepared by dissolving in 200 μl PBS to the desired concentration of the sample for 7 days and administered orally by 200 μl (normal group and control group administered orally 200 μl, using promethazine hydrochloride as a positive control). The visual evaluation scores were measured once a week, and in FIG. 18, ear edema was measured.

In addition, the treatment effect by measuring the weight of the spleen and ears at the time of sacrifice is shown graphically in Figures 19 and 20, blood was collected at the time of sacrifice to measure the number of leukocytes, eosinophils, basophils and graphically shown in Figures 21 to 23. . And at the time of sacrifice, the back tissue was taken, crushed into lysis buffer, and inflammatory cytokine (TNF-α, IFN-γ, IL-17) was measured using an ELISA kit (ebioscience Com. USA). The results are shown in FIGS. 24 to 26. Significance was indicated by t-test statistical treatment (# = p <0.05 compared with normal group, * = p <0.05 compared with control group). In FIG. 23, the actinigen content of the gall bladder extract was 71%.

As a result of visual measurement of DNCB atopy animal experiments, as shown in FIG. 17, inflammation, bleeding, swelling, and molting intensified in the 2nd and 3rd weeks of disease induction in all groups except the normal group. After 3 weeks, each sample was administered orally once a day for a total of 7 days, significantly higher than the control group, 95% actinizenin (as burdock extract tablet concentrate) 50 mg / kg administration group, Inflammation, bleeding, edema, and hair loss were recovered in the 98% tannic acid 50 mg / kg group, the complex AT101 20 mg / kg group, the complex AT101 50 mg / kg group, and the positive control promethazine 1 mg / kg group. .

As a result of DNCB atopy animal test ear thickness measurement, as shown in FIG. 18, in the atopic derive group except the normal group, the ear thickness becomes thick due to edema over the 2nd and 3rd weeks of disease induction. After 3 weeks, each sample was orally administered once a day for a total of 7 days, and 95% actigenin (as burdock extract tablet concentrate) 20 mg / kg group, 95% significantly compared to the control group. Actigenin (as burdock extract tablet concentrate) 50 mg / kg administration group, 98% tannic acid 50 mg / kg administration group, complex AT101 20 mg / kg administration group, complex AT101 50 mg / kg administration group, positive control promethazine 1 mg / Ear edema was recovered in the kg-treated group.

In the DNCB atopic animal experiment, the weight of the spleen, which is an immune organ, and the weight of the ear (Fig. 20), the weight of the spleen was significantly increased due to the immune system activity in the control group, compared to the normal group. The weight of the ear increased significantly compared to the normal group. On the other hand, each sample was administered orally once a day for a total of seven days, compared with the control group significantly 95% actinigen (as burdock extract tablet concentrate) 50 mg / kg administration group, 98% tannic acid 50 mg / kg administration group , AT101 50mg / kg complex, promethazine 1mg / kg positive control group stabilized immune system, reduced spleen weight, ear edema was recovered, ear weight was reduced.

As a result of DNCB atopic animal test blood analysis, as shown in Figure 21 to Figure 23, the number of leukocytes, eosinophils, basophils due to the immune system activity in the control group significantly increased compared to the normal group. On the other hand, each sample was orally administered once a day for a total of 7 days, compared with the control group significantly 95% actinigenin (as burdock extract tablet concentrate) 50 mg / kg administration group, complex AT101 50 mg / kg administration group, In the positive control promethazine 1 mg / kg group, the immune system was stabilized and the number of leukocytes, eosinophils, and basophils was reduced.

As a result of DNCB atopic animal experiment tissue inflammation cytokine analysis, as shown in Figure 24 to 26, the expression of inflammatory cytokines (TNF-α, IFN-γ, IL-17) due to the immune system activity in the control group and the normal group Significantly increased in comparison. 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 complex AT101 50 mg / kg administration group, resulting in the expression of inflammatory cytokines (TNF-α, IFN-γ, IL-17). All showed a decrease.

As described and demonstrated in detail above, the present invention provides a natural product extract complex (AT101) comprising actinogen and tannic acid as an active ingredient having anti-inflammatory, histamine release and allergic disease alleviating effects. AT101 inhibits 5-lipoxygenase (5-LO) and cyclooxygenase (COX-2) and inhibits the expression of inflammatory cytokines (TNF-α, IL-1β, IL-6) It was confirmed that there is an excellent suppression of the release of histamine, the most important mechanism in allergic diseases. Animal studies have shown that type 1 disease may be reduced by attenuating passive skin anaphylaxis by inhibiting Evans blue leakage, leukotriene production, and prostaglandin E ₂ production. As well as improving the allergic reaction, it showed a similar level of anti-allergic function as the positive control promethazine, it was confirmed that it can act as a composition that can effectively improve allergic diseases.

Although the present invention has been described in detail only with respect to the described embodiments, it will be apparent to those skilled in the art that various modifications and variations are possible within the technical scope of the present invention, and such modifications and modifications are within the scope of the appended claims.

N: normal group C: control group
A1: Burdock Extract (Actigenin 0.315%) A2: Burdock Extract (Actininin 43.24%)
A3: Burdock Extract (Actininin 95.00%)
T1: 40% tannic acid T2: 70% tannic acid
T3: 98% tannic acid
AT1: 95% Actinigen (as Burdock Extract Purified Concentrate): 98% Tannic Acid = 2: 1
AT2: 95% actinigen (as burdock extract purified concentrate): 98% tannic acid = 4: 1
AT3: 95% actinigen (as burdock extract purified concentrate): 98% tannic acid = 5: 1
AT4: 95% actinigen (as burdock extract purified concentrate): 98% tannic acid = 8: 1
P: promethazine

Claims (9)

Containing burdock extract and tannic acid as an active ingredient, burdock extract: tannin acid = 4.1: 1 to 7.9: 1 by weight ratio composition for allergic diseases relief comprising. delete The method of claim 1,
A composition for allergy disease alleviation, characterized in that the burdock extract contains 95% actinigen. delete A dietary supplement for allergy disease alleviation, comprising the composition of claim 1. The method of claim 5,
A dietary supplement for allergy disease alleviation, comprising the composition according to claim 1 in an amount of 0.1 to 100% by weight. The method of claim 6,
A dietary supplement for allergy disorder alleviation of allergic diseases wherein the dietary supplement is a tablet, capsule, pill or liquid. A coating agent for alleviating allergic diseases comprising the composition of claim 1. The coating agent for alleviating allergic diseases according to claim 8, comprising the composition according to claim 1 in an amount of 0.1 to 100% by weight.

Images