KR20120121684A - Antipruritic composition containing astragalin and quercetin - Google Patents

Abstract

PURPOSE: An antipruritic composition containing quercetin and astragalin is provided to ensure suppress and relieve skin itching and to relieve atopic dermatitis. CONSTITUTION: An external use skin composition for treating atopic dermatitis contains 0.001-10 wt% of each quercetin of chemical formula 1 and astragalin of chemical formula 2. Qeurcetin is a natural compound belonging to polyphenol flavonoid of fruits, vegetables, and various plants. Astragalin is a flavonoid glycoside compound contained in plant leaves including persimmon leaves. The composition is manufactured in the form of a pharmaceutical compositon of a cream, essence, lotion, ointment, paste, liniment, external use liquid, aerosol, and plaster. [Reference numerals] (AA) Structure of 3.3' .4' .5.7-pentahydroxy flavone(quercetin); (BB) Structure of kampferol-3-0-glucopyranoside(astragalin)

Description

Anti-itch composition containing quercetin and astragalin {Antipruritic composition containing astragalin and quercetin}

The present invention relates to an anti-itch composition containing quercetin and astragalin, and more particularly, to provide anti-inflammatory effects by simultaneously using quercetin and astragalin, and to suppress and improve the itching of the skin to relieve atopic dermatitis. It relates to an external preparation for skin and an oral composition.

This study was supported by the Small and Medium Business Administration and Jeollabuk-do. This study was also supported by Jeonju University.

Atopic dermatitis (AD) is a chronic disease with aggravation and remission and severe itching. It is a skin inflammatory disease caused by multifactorial factors such as immunological dysfunction and exposure to allergens. In particular, itching, which is a major symptom of atopic dermatitis, is caused by skin lesions and severe psychological disturbances, and is one of the biggest causes of worsening the quality of life of patients with atopic dermatitis. In addition, scratching the affected area due to itching causes the breakdown of the skin barrier, and the repeated inflammation of the skin worsens due to repetitive itching. Therefore, effective control of the itch is considered important in the treatment of atopic dermatitis.

Mast cells are the major cells that produce inflammatory disease mediators such as allergies or acute allergic reactions. Acute allergic reactions are caused by histamine secretion as the antigen bound to IgE binds to the FcεRI receptor in mast cells. After activation of FcεRI, mast cells begin the degranulation process as a result of secretion of mediators such as arachidonic acid metabolites and inflammatory cytokines. Among inflammatory substances secreted from mast cells, histamine is the most characteristic and strong vascular mediator and is a substance causing hypersensitivity. Mast cells activated by stimulation of various antigens include histamine, inflammatory mediators such as eicosanoid, proteoglycan, protease, and pro-inflammatory cytokine and chemotactic cytokines. Produces cheinactic cytokine. Strongly stimulating mast cells are known to cause itching of substances such as compound 48/80 (compound 48/80) and basic amino acid complexes such as substance P (substance P). In particular, compounds 48/80 Is known as a direct and convenient substance for the study of the mechanism of anaphylactic allergic reaction.

The present inventors have attempted to provide a substance derived from natural products that can suppress and improve skin itching, which is a major symptom of atopic dermatitis. Through this study, a combination of astragalin and quercetin was used to provide excellent anti-inflammatory effect and compound 48/80. The present invention has been accomplished by discovering that it is possible to suppress and improve skin itching induced by histamine, serotonin or substance P.

Accordingly, an object of the present invention is to provide a composition that can alleviate atopic dermatitis using quercetin and astragalin.

In order to achieve the above object, the present invention provides a topical external preparation and oral composition for improving atopic dermatitis containing quercetin and astragalin.

The composition of the present invention can be effectively alleviated atopic dermatitis by providing an excellent anti-inflammatory effect and itching inhibition and improvement by containing quercetin and astragalin.

1 is a graph showing the effect of quercetin and astragalin on the inhibition of histamine release of KU812 cells. QT stands for quercetin, AG stands for astragalin, ^# p <0.001 represents a comparison with the non-drug treated negative control, ^* p <0.05 and ^** p <0.01 are positive treated with anti-FcεRIα antibody only Comparison with the control is shown.
2 is a graph showing the synergistic effect of quercetin and astragalin on the inhibition of histamine release of KU812 cells. QT stands for quercetin, AG stands for astragalin, ^# p <0.001 represents a comparison with the non-drug negative control, and ^* p <0.05 and ^*** p <0.001 were treated with anti-FcεRIα antibodies only. Comparison with positive control is shown.
Figure 3 is a graph showing the effect of quercetin and astragalin on the inhibition of nitric oxide production of peritoneal macrophages. QT stands for quercetin, AG stands for astragalin, ^# p <0.001 represents a comparison with the negative control without drug treatment, and ^* p <0.05 and ^** p <0.01 for the positive control treated with LPS only. The comparison is shown.
4 is a graph showing the synergistic effect of quercetin and astragalin on the inhibition of nitric oxide production of peritoneal macrophages. QT stands for quercetin, AG stands for astragalin, ^# p <0.001 represents a comparison with the negative control without drug treatment, and ^* p <0.05 and ^*** p <0.001 for the positive control treated with LPS only. Indicates a comparison.
5 is a graph showing the effects of quercetin and astragalin on the inhibition of prostaglandin E ₂ production in abdominal macrophages. QT stands for quercetin, AG stands for astragalin, ^# p <0.001 represents a comparison with the negative control without drug treatment, and ^* p <0.05 and ^** p <0.01 for the positive control treated with LPS only. The comparison is shown.
6 is a graph showing the synergistic effect of quercetin and astragalin on the inhibition of prostaglandin E ₂ production in celiac macrophages. QT stands for quercetin, AG stands for astragalin, ^# p <0.001 represents a comparison with the negative control without drug treatment, and ^* p <0.05 and ^*** p <0.001 for the positive control treated with LPS only. Indicates a comparison.
7 is a graph showing the synergistic effect of quercetin and astragalin on the inhibition of Th2 cytokine production in splenocytes. QT stands for quercetin, AG stands for astragalin, ^# p <0.001 represents a comparison with the untreated drug negative control, ^* p <0.05 and ^*** p <0.001 for anti-CD3 and anti-CD28S Only comparison with the positive control group treated.
FIG. 8 is a graph showing the synergistic effect of quercetin and astragalin on itch relief induced by compound 48/80. QT stands for quercetin, AG stands for astragalin, ^# p <0.001 represents a comparison with the untreated drug-negative control, and ^* p <0.05 and ^*** p <0.001 were treated with Compound 48/80 only Comparison with positive control is shown.
9 is a graph showing the synergistic effect of quercetin and astragalin on the relief of itching induced by histamine. QT stands for quercetin, AG stands for astragalin, ^# p <0.001 represents a comparison with the negative control without drug treatment, and ^* p <0.05 and ^*** p <0.001 for the positive control treated with histamine only. Indicates a comparison.
10 is a graph showing the synergistic effect of quercetin and astragalin on the relief of itching induced by serotonin. QT stands for quercetin, AG stands for astragalin, ^# p <0.001 represents a comparison with the negative control without drug treatment, and ^* p <0.05 and ^*** p <0.001 for the positive control treated with serotonin only. Indicates a comparison.
FIG. 11 is a graph showing the synergistic effect of quercetin and astragalin on the relief of itching induced by substance P. QT stands for quercetin, AG stands for astragalin, ^# p <0.001 represents a comparison with the negative control without drug treatment, and ^* p <0.05 and ^*** p <0.001 represent a positive control treated with substance P only. The comparison with.

The composition of the present invention relates to a composition containing quercetin and astragalin, specifically, to improve atopic dermatitis that inhibits and improves the anti-inflammatory effect and skin itching induced by compound 48/80, histamine, serotonin or substance P, etc. It relates to an external preparation for skin or an oral composition.

Quercetin (quercetin) used in the present invention is a natural compound belonging to the polyphenolic flavonoid (polyphenolic flavonoid) contained in abundance in fruits, vegetables, various plants, and inhibits and anti-allergic to antioxidant, anticancer, antiviral, cardiovascular diseases It is a substance well known to exhibit various bioactive effects. The structure of quercetin is shown in the following formula (1).

On the other hand, astragalin, which is used as an active ingredient in the present invention, is a flavonoid glycoside compound mainly contained in various parts including leaves of plants including persimmon leaves, and recently, bioactive effects such as antioxidants have been reported. The structure of astragalin is shown in the following formula (2).

The composition according to the present invention contains quercetin and astragalin in an amount of 0.001 to 10% by weight, respectively, based on the total weight of the composition. In addition, in the composition of the present invention, the quercetin and astragalin are combined in a ratio of 1: 1.

The composition of the present invention may be a topical skin composition or an oral composition. In addition, the topical skin composition may be a cosmetic composition or a pharmaceutical composition, and the oral composition may be a food composition or a pharmaceutical composition. It may also be a parenteral composition such as an injection.

Although the formulation is not specifically limited in the composition of this invention, In the case of the external application composition for skin, cosmetics, such as a cream, an essence, a lotion; Or in the form of pharmaceutical compositions such as ointments, creams, pastes, lotions, liniments, external solutions, tinctures, aerosols, plasters and the like. In addition, the compositions of the present invention may be formulated as pharmaceutical or food compositions in the form of pills, capsules, sweets, granules, drinks or injections.

The compositions of the present invention can be prepared in pharmaceutical formulations according to conventional methods. In the preparation of the formulations, it is preferred that the active ingredient is mixed with or diluted with the carrier, or enclosed in a carrier in the form of a container. When the carrier is used as a diluent, it may be a solid, semisolid or liquid substance which acts as a carrier, excipient or medium for the active ingredient. Thus, the formulations may be in the form of tablets, pills, powders, assays, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, sterile injectables, sterile powders and the like.

Examples of suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydride Oxybenzoate, talc, magnesium stearate and mineral oil. The formulations may additionally include fillers, anti-coagulants, lubricants, wetting agents, perfumes, emulsifiers, preservatives, and the like. Compositions of the present invention may be formulated using methods well known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.

The pharmaceutical composition of the present invention may be administered through several routes including oral, transdermal, subcutaneous, intravenous, abdominal, muscle, topical application, patch and iontophoresis, of which topical application and oral administration are preferred. Do.

In addition, the range of prophylactic or therapeutic doses for atopic dermatitis in the composition according to the present invention may vary depending on the severity and dosage form, and the number of applications may also vary depending on the age, weight and constitution of the patient.

For example, the dosage of the external preparation composition for skin of the present invention may vary depending on age, sex, weight, symptoms, and method of administration, but it is 1.0 to 3.0 ml per day, which is applied 1 to 5 times a day to continue for more than 1 month. It is good.

In addition, for example, a typical daily dosage of an oral composition of the present invention may range from 1 to 100 mg / kg body weight, preferably 5 to 70 mg / kg body weight, to be administered once or in several divided doses. Can be. However, the dosage does not limit the scope of the invention in any way.

Hereinafter, the present invention will be described in more detail with reference to Examples and Test Examples, but the present invention is not limited only to these examples.

Reference Example 1 Isolation and Identification of Quercetin and Astragaline

The present invention was used to separate the quercetin and astragalin from the mulberry ( Morus alba ) leaves (upper side) and the specific separation method is as follows. In addition, mulberry tree identification was identified at the Department of Herbal Control, College of Oriental Medicine, Woosuk University.

1. Isolation of Bioactive Substances from Mulberry Leaves

Mulberry leaves were collected from the mulberry tree on June 3, 2010 in Weebong-ri, Daeheung-ri, Soyang-myeon, Wanju-gun, Jeollabuk-do. The collected mulberry leaves were immediately washed with fresh water, dried at 45 ° C. for 12 hours, and then chopped and 530g were repeatedly extracted three times at 50 ° C. with methanol using an extractor for 3 hours. The extract was concentrated under reduced pressure in an aqueous phase to obtain about 67 g of methanol extract. The methanol extract was suspended in distilled water, and the solvent was fractionated in the order of n -hexane, methylene chloride, ethyl acetate and n -butanol in the order of 13.2 g, 1.48 g, 1.32 g, and 6.31 g of fraction were obtained.

1.2 g of an ethyl acetate fraction was subjected to column chromatography (35 × 3.0 cm) with Sephadex LH-20 and methanol, followed by TLC (silica gel 60F ₂₅₄ , developing solvent; toluene: ethyl acetate: formic acid = 5 :). 4: 1, v / v ) divided into six fractions (E1-E6) according to the pattern.

Of these, material separation was attempted at E4, which shows a major spot. E4 (467 mg) was partitioned into 8 small fractions (E41-E48) by column chromatography (17 x 2.5 cm) with a mixed solvent of silica gel and CHCl ₃ : MeOH: H ₂ O (90: 20: 1). . Among them, E44 (23 mg) was subjected to preparative high-speed chromatography (prep-HPLC) using a GS310 column and MeOH to obtain Compound 1 (8.4 mg). Preparative high-speed chromatography (prep-HPLC) was performed on a GS310 column and MeOH using E42 (78 mg) to obtain Compound 2 (18.7 mg).

2. Structure Identification of Quercetin and Astragaline

Compound 1 first showed positive (+) in the Molish test and the flavonoid color reaction FeCl ₃ , Mg-HCl, Zn-HCl. Δ 7.74 (1H, d, J = 2.4 Hz), δ 6.87 (1H, d, J = 2.4 Hz), δ 6.87 (1H, d, J = 8.6 Hz) and δ 7.64 in the aromatic region of the ¹ H-NMR spectrum (1H, dd, J = 8.6, 2.4 Hz) is a peak attributable to H-2 ', H-5' and H-6 'of the B ring of Chemical Formula 1, where H-5' is adjacent to hydrogen (proton) It was observed that meta ( J = 2.4 Hz) and ortho coupling ( J = 8.8 Hz) each had two proton peaks corresponding to 2H. As a result, it was confirmed that the structure of the ring B is a catechol structure having an ABX system structure. In addition, since no signal of hydrogen related to the C ring was observed, it was estimated to be a flavonoid compound, and signals corresponding to C-3 and C-2, which are characteristic of flavonoid compounds at δ 136.1 and 146.8 of ¹³ C-NMR spectra, respectively. Was observed. In addition, it was found that δ 147.6 and δ 145.1 have a catechol structure in which OH (hydroxyl group) is bonded to C-4 ′ and C-3 ′ of the B ring. C-8 and C-6 of ring A are shown at δ 94.1 and δ 98.1 respectively, and C-5, C-7 and C-9 with OH (hydroxyl) bonds are found at 161.3, 164.4 and 157.1, respectively, and typical phloroglucinol (phloroglucinol) A ring was confirmed.

Results and literature [Jin Eun-young, Park Young-seo, Jang Jae-kwon, Jung Myeong-su, Park Hoon, Shim Gun-seop, Choi Young-jin. Extraction of Quercetin-Related Substances from Onion Pulp by Solvent Extraction and Combined Extraction, 2009, Industrial Food Engineering, 13 (2), 147-153], Compound 2 was 3,3 ', 4', 5,7- The structure was identified as quercetin, a pentahydroxyflavone.

3,3 ', 4', 5,7-pentahydroxyflobon (quecetin) : ¹ H-NMR (400 MHz, CD ₃ OD) δ 6.19 (1H, d, J = 2.4 Hz, H-6), 6.39 (1H, d, J = 2.4 Hz, H-8), 6.87 (1H, d, J = 8.6 Hz, H-5 '), 7.63 (1H, dd, J = 8.6, 2.4 Hz, H-6' ), 7.74 (1H, doublet, J = 2.4 Hz, H-2 '). ¹³ C-NMR (100 MHz, CD ₃ OD) δ 176.5 (C-4), 164.4 (C-7), 161.3 (C-5), 157.1 (C-9), 147.6 (C-4 '), 146.8 ( C-2), 145.1 (C-3 '), 136.1 (C-3), 122.9 (C-1'), 120.5 (C-6 '), 115.1 (C-5'), 114.8 (C-2 ' ), 103.3 (C-10), 98.1 (C-6), 94.1 (C-8).

Compound 2 was positive for FeCl ₃ , Mg-HCl and Zn-HCl, Molish test and flavonoid color reaction. ^In the aromatic region of the ¹ H-NMR spectrum, two proton peaks corresponding to 2H were observed, ortho coupling ( J = 8.8 Hz) at δ 8.04 and 6.88, and meta-coupling at δ 6.38 and 6.19. A signal corresponding to 1H of meta coupling (J = 2.0 Hz) was observed.

As a result, Compound 2 was assumed to be a flavonoid compound which is a camphorol skeleton, and a peak estimated as anomer proton ( J = 7.6 Hz) was observed at δ 5.24, and δ corresponding to glucose in ¹³ C-NMR spectrum. Five of 104. 1 (C-1 "), 78.4 (C-3"), 87.1 (C-5 "), 75.7 (C-2"), 71.4 (C-4 "), including a methylene peak of 62.6 Peaks were observed.

The structure of the compound 2 is estimated by the camphorol glycosides as described above [Do JC, Yu YJ, Jung KY, and Son KH. (1992a) Flavonoids from the leaves of Polygala japonica . Kor. J. Pharmacogn. 23. 9-13 and Do JC, Jung KY, and Son KH. (1992b) Flavonoid glycosides from the frods of Pyrrosia lingua . Kor. J. Pharmacogn. 23. 276-269 was confirmed as camphorol-3-O-glucopyranoside (astragalin) in comparison with the data published.

Camperol-3-O-glucopyranoside (astragalin) : ¹ H-NMR (400 MHz, CD ₃ OD) δ 8.04 (2H, d, J = 8.8 Hz, H-2 ', 6'), 6.88 ( 2H, d, J = 8.8 Hz, H-3 ', 5'), 6.38 (1H, d, J = 2.0 Hz, H-8), 6.19 (1H, d, J = 2.0 Hz, H-6), 5.24 (1H, doublet, J = 7.6 Hz, H-1 "). ¹³ C-NMR (100 MHz, CD ₃ OD) δ 179.5 (C-4), 166.3 (C-7), 163.1 (C-5), 161.6 (C-4 '), 159.1 (C-9), 158.5 (C-6), 135.5 (C-7), 132.3 (C-2', 6 '), 122.8 (C-1'), 116.1 ( C-3 ', 5'), 105.7 (C-10), 104.1 (C-1 "), 100.0 (C-6), 94.8 (C-8), 78.4 (C-3"), 87.1 ( C-5 "), 75.7 (C-2"), 71.4 (C-4 "), 62.6 (C-6").

Reference Example 2 Materials

1. Reagent

Enzyme-Linked ImmunoSorbent Assay (ELISA) kits such as Prostaglandin E ₂ , PGE ₂ , Interleukin-4 (IL-4), IL-13, Immunoglobulin E (IgE) and anti mouse CD3, anti mouse CD28 Was purchased from R & D Systems (Minneapolis, USA). RPMI 1640 medium, fetal bovine serum (FBS) and penicillin / streptomycin were purchased from Gibco BRL (Grand Islang, NY, USA). In addition, Lipopolysacchride (LPS), Compound 48/80, Histamine, Serotonin, Substance P, N G-Monomethyl-Larginine (NGMMA) and all other reagents used are Sigma-Aldrich and Merck as analytical grades. (Darmstadt, Germany).

2. Experimental Animal

Six-week-old female hairless mice were purchased from Orient Bio Inc. (Seoul, South Korea) and used for experiments after adapting to the environment for one week after purchase. Mice were fed freely with feed (Central Experimental Animal Co., Ltd.) and sterilized water by fixing the day and night cycles for 12 hours, and maintaining the temperature and humidity at 22 ± 1 ℃ and 60 ± 5%, respectively. It was used for the experiment in accordance with the experimental regulations of the University Animal Experiment Committee.

Reference Example 3 Statistical Processing

All experimental values were expressed as mean ± standard error, statistical analysis was performed by ANOVA and student's t-test, and the significance limit was set at p <0.05.

Test Example 1 Measurement of Histamine Release Inhibitory Effect

KU812 cells (human basophilic leukemia cells) were cultured in RPMI 1640 medium containing 10% FBS with sufficient moisture and 37 ° C. in the presence of 95% air and 5% CO ₂ . KU812 cells were injected with histamine release buffer (30 mmol / L TRIS-HCl [pH 7.6], 120 mmol / L NaCl, 5 mmol / L KCl, 1 mmol / L CaCl ₂ , 1 mmol / L MgCl ₂ , and 0.03% BSA). The first culture of 100 μL of KU812 cells (10 ⁵ / mL) was Quecetin (5, 10, 20 μg / mL), Astragalin (5, 10, 20 μg / mL), or Quecetin (5 μg / mL) and Astragalin (5 μg). / mL) were injected at the same time and left in an ice bath for 30 minutes. Then, centrifuged at 3,000 rpm for 5 minutes at 4 ℃, 200μL injection of histamine release buffer, anti-FcεRIα mAb (15μg / mL) was injected and incubated at 37 ℃ for 30 minutes. Cell suspension was then centrifuged at 3,000 rpm for 5 minutes to separate supernatant and cells, and cell eluate was cell lysis buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na ₂ EDTA, 1 mM EGTA (ethylene glycol tetraacetic acid, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na ₃ VO ₄ , 1 μg / ml leupeptin The cells were lysed and the supernatant and cell eluate were measured using a histamine ELISA assay kit. The normal control group measured the amount of histamine not stimulated with anti-FcεRIα mAb, and the positive control group measured the amount of histamine stimulated with anti-FcεRIα mAb. The measured results are shown in FIGS. 1 and 2.

1, it can be seen that quercetin and astragalin have an effect of inhibiting histamine release depending on concentration. In particular, both quercetin and astragalin were treated with a concentration of 10 μg / mL and 20 μg / mL, but the histamine release inhibitory effect was weak in the low concentration of 5 μg / mL treatment group.

However, as can be seen in Figure 2, when the low concentration (5μg / mL) of the quercetin and astragaline at the same time treatment having no significant effect on the inhibition of histamine release to make the final treatment concentration of the active material 10μg / mL, It can be seen that there is a remarkably superior histamine release inhibitory effect than when quercetin or astragalin is treated at a concentration of 10 μg / mL, respectively.

This results in synergistic effects by treating quercetin and astragalin at the same time, and more effectively controlling the release of histamine associated with skin itch.

Test Example 2 Measurement of Nitric Oxide Inhibitory Effect

1. Isolation and Culture of Peritoneal Macrophages

Healthy male mice were injected intraperitoneally with 2 mL of thioglycolate broth (Brewer's thioglycollate broth) using a syringe, and after 3 days, mice were immediately killed by pedicle decay and 10 mL of HBSS to which heparin (5U / mL) was added was intraperitoneally. And the abdominal cavity was well massaged. After the massage, cells were introduced into the abdominal cavity using a 26-cage 10 mL syringe and centrifuged twice. The isolated cells were suspended in RPMI 1640 medium containing 10% FBS and 1% penicillin / streptomycin and inoculated in a culture plate, and then left for 2 hours in an incubator maintained at 37 ° C. and 5% CO ₂ . Thereafter, the cells were washed three times using cold HBSS to remove other cells. Attached mouse intraperitoneal macrophages were inoculated in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin / streptomycin and cultured for use in the following experiments.

2. Measurement of Nitric Oxide Concentration

L-arginine, a substrate of nitric oxide (NO), turns into L-citrulline and nitrogen monoxide, which quickly turn into stable nitrogen dioxide, nitrite and nitrate. The grease reagent (0.5% sulfanylamide, 2.5% phosphoric acid and 0.5% naphthylethyleneamine) chemically reacts with nitrite to form a purple azo salt, which matches the concentration of nitrogen monoxide. The concentration of nitrite can be determined from the salt concentration, which can be found by measuring its maximum absorption at 540 nm.

To investigate the effect of quercetin and astragalin on the nitric oxide production ability of peritoneal macrophages isolated from mice, the peritoneal macrophages (5 × 10 ⁵ / mL) isolated from the mice were treated with quercetin (5, 10, 20 μg). / mL), Astragalin (5, 10, 20μg / mL), or Quecetin (5μg / mL) and Astragalin (5μg / mL) were treated simultaneously, and also NGMMA (PGE ₂ production inhibitor, known as a control) 5 μM) was used. After the various concentrations of the drug were pretreated, and after 2 hours, washed three times with RPMI 1640 medium, and then stimulated with LPS (1 μg / mL) and incubated for 24 hours. The culture supernatant was obtained and the amount of nitrogen monoxide produced was measured. That is, 100 μL of the cell supernatant of each of the control group and the experimental group was injected into a 96-well plate, and 100 μL grease reagent was added to react for 30 minutes at room temperature. The absorption of the sample light was measured at 540 nm with a spectrophotometer (MD, USA). The degree of nitrite concentration was calculated from the standard curve of nitrite. The results are shown in FIGS. 3 and 4.

Referring to Figure 3, it can be seen that in the peritoneal macrophages stimulated by LPS, quercetin and astragalin have an effect of inhibiting nitrogen monoxide production depending on the concentration. In particular, when both quercetin and astragalin were treated at concentrations of 10 μg / mL and 20 μg / mL, there was a significant effect of inhibiting nitric oxide production, but the low concentration of 5μg / mL treatment group showed a weak effect of inhibiting nitric oxide production. have.

However, as can be seen in Figure 4, when the low concentration (5μg / mL) of the quercetin and astragaline treatment at the same time having no significant effect on the inhibition of nitric oxide production when the final treatment concentration of the active material is 10μg / mL It can be seen that quercetin or astragalin has a significantly superior effect of inhibiting the production of nitric oxide than when treated at a concentration of 10 μg / mL, respectively. In addition, it can be confirmed that this is similar to the effect of NGMMA, which is well known as a nitric oxide production inhibitor.

Through this, the treatment of quercetin and astragalin at the same time can be synergistic effect, it can be seen that more effectively regulate the production of nitric oxide associated with skin itching.

Test Example 3 Measurement of Prostaglandin E2 Inhibitory Effect

1. Isolation and Culture of Peritoneal Macrophages

Healthy male mice were injected intraperitoneally with 2 mL of thioglycolate broth (Brewer's thioglycollate broth) using a syringe, and after 3 days, mice were immediately killed by pedicle decay and 10 mL of HBSS to which heparin (5U / mL) was added was intraperitoneally. And the abdominal cavity was well massaged. After the massage, cells were introduced into the abdominal cavity using a 26-cage 10 mL syringe and centrifuged twice. The isolated cells were suspended in RPMI 1640 medium containing 10% FBS and 1% penicillin / streptomycin and inoculated in a culture plate, and then left for 2 hours in an incubator maintained at 37 ° C. and 5% CO ₂ . Thereafter, the cells were washed three times using cold HBSS to remove other cells. Attached mouse intraperitoneal macrophages were inoculated in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin / streptomycin and cultured for use in the following experiments.

2. Determination of Activity of Prostaglandin E ₂ (PGE ₂ )

Peritoneal macrophages (5 × 10 ⁵ / mL) isolated as described above were treated with quercetin (10 μg / mL), astragalin (10 μg / mL), or quercetin (5, 10, 20 μg / mL), or astragalin ( 5, 10, 20 μg / mL, or quercetin (5 μg / mL) and astragalin (5 μg / mL) were treated simultaneously, and also treated with indomethacin (5 μM), a PGE ₂ production inhibitor known as a control. Was used. The various concentrations of the drugs were pretreated and stimulated with LPS (100 ng / mL) after 2 hours, followed by incubation for 24 hours. Culture supernatants were obtained and the amount of prostaglandin E ₂ was measured. Quantification of these materials measured the amount accumulated over 24 hours. The measurement method of prostaglandin E ₂ was quantified by ELISA method according to the method proposed by R & D (Minneopolis, USA). The results are shown in FIGS. 5 and 6.

5, it can be seen that in the LPS-stimulated peritoneal macrophages, quercetin and astragalin have an effect of inhibiting the production of prostaglandin E ₂ depending on the concentration. In particular, both quercetin and astragalin had significant inhibitory effects on prostaglandin E ₂ production when treated at concentrations of 10 μg / mL and 20 μg / mL, but the inhibitory effect of prostaglandin E ₂ production was weak in the low concentration of 5 μg / mL. You can check it.

However, as can be seen in Figure 6, the low concentration (5μg / mL) of the quercetin and astragaline treatment without a significant effect on the inhibition of prostaglandin E ₂ production at the same time to the final treatment concentration of the active material to 10μg / mL In this case, it can be seen that the prostaglandin E ₂ production inhibitory effect is remarkably superior to that of quercetin or astragalin at the concentration of 10 μg / mL, respectively. In addition, it can be confirmed that this is similar to the effect of indomethacin (indomethacin) well known as prostaglandin E ₂ production inhibitor.

It can be seen that synergistic effects can be obtained by treating quercetin and astragalin simultaneously, thereby more effectively controlling the production of prostaglandin E ₂ associated with skin itch.

Test Example 4 Effect of Th2 Cytokine Inhibition

1. Isolation of Splenocytes

Healthy male mice were anesthetized with ether and immediately killed by posterior tibial vertebral debridement to separate splenocytes. Gently squeezed with slide glass to create a single cell suspension and then passed through a 40㎛ nylon mesh. Cells were centrifuged twice using HBSS without Ca ²⁺ and Mg ²⁺ . Thereafter, lymphocytes were isolated using Ficoll-hypaque and centrifuged three times with RPMI 1640 culture. The cells were suspended in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin / streptomycin, followed by confirmation of cell viability of 95% or more with 0.4% trypan blue, and counting the number of cells using a hemocytometer slide. It was used for the following experiment while culturing.

2. Measurement of IL-4, IL-5 and IL-13 Cytokines in Splenocyte Supernatants

Splenocytes (2.5 × 10 ⁶ / mL) were treated with quercetin (10 μg / mL), astragalin (10 μg / mL), or quercetin (5 μg / mL) and astragalin (5 μg / mL), and also as a control. A treatment with Tacrorimus (5 μM), a known PGE ₂ production inhibitor, was used. After two hours of pretreatment of the various concentrations of the above-described drugs, stimulation was simultaneously performed with anti-mouse CD3 (1 μg / mL) and anti-mouse CD28 (1 μg / mL), and 48 hours later, culture supernatants were obtained. The amount of IL-13 cytokine was measured. Cytokines were measured by ELISA according to the method suggested by R & D (Minneopolis, USA). The results are shown in Fig.

Referring to FIG. 7, the experimental group treated with 10 μg / ml of quercetin and istragaline, respectively, showed an effect of inhibiting IL-4 and IL-13 (p <0.05), in particular, quercetin and astra In the experimental group treated with gallin at the same time, it can be seen that the synergistic effect of these components significantly reduced the production of IL-4 and IL-13. In addition, it can be confirmed that this is similar to the effect of Tacrorimus (Tacrorimus) well known as a Th2 cytokine inhibitor.

Allergic skin diseases such as atopy generally have immunological characteristics in which Th2 cells are more biased than Th1 cells, and cytokines such as IL-4 and IL-13 are produced very high, and B cells are activated. This results in excessive secretion of IgE antibodies that cause allergic diseases. Therefore, the discovery of a substance that effectively inhibits Th2 cytokines such as IL-4 and IL-13 is a great help in improving skin diseases causing itching.

Therefore, as confirmed in the present experiment, it can be seen that the use of the combination of quercetin and astragalin, respectively, rather than the use alone in combination has a greater effect in treating or improving the skin disease causing itching.

Test Example 5 Measurement of Itching Reduction Effect on Itching Inducing Material

Before starting the experiment, five hairless mice (7 weeks old) per experimental group were placed in a transparent acrylic cage (20 × 26 × 13 cm), respectively, and left in the same experimental environment for 30 minutes for stability. Prior to causing itching, quercetin (10 mg / kg (body weight)) or astragalin (10 mg / kg (body weight)), or quercetin (5 mg / kg) and astragalin (5 mg / kg) are administered orally simultaneously and controls 0.20 ml of silver phosphate buffer solution was orally left for 1 hour. Itching compounds 48/80, histamine, serotonin, and substance P were then dissolved or diluted in physiological saline, respectively, and injected sc into the height between the shoulders of the mice. At this time, compound 48/80 50μg / site, histamine 100μg / site, serotonin 100μg / site, substance P 100μg / site concentration according to each experimental group was used 0.10μl. Mice injected with an itch-inducing substance were immediately followed by a microcamera following the method of Mihara (Kuraishi et al. , Scratching behavior induced by pruritogenic but not algesiogenic agents in mice.Eur J Pharmacol ., 275 : 229-223, 1995). 60 minutes were recorded using a camera (ONCCTV, Seoul, South Korea), and the number of scratches on the site where the itch-inducing substance was injected using the hind paw was evaluated by counting the double blind method. The experiments for each trigger were conducted on different days and mice used in each experiment were used once. The measurement results are shown in FIGS. 8 to 11.

As shown in FIGS. 8 to 11, the number of scratches due to the itch caused by Compound 48/80, histamine, serotonin, substance P, and the like, was compared with the test group administered with quercetin and astragalin, respectively. It is much less at, and it can be confirmed that the synergistic effect of the simultaneous use of quercetin and astragalin to reduce the itching effect. In addition, it can be seen that it is superior to the effect of the drug metysergide (methysergide) used to reduce the itch.

Therefore, in order to show the effect on the itch, it is possible to obtain a much better effect by synergistic effect of the administration of the combination of these at the same time than using quercetin and arthralgarine alone.

Claims (8)

A topical skin composition for improving atopic dermatitis containing quercetin and astragalin. The composition for external application for skin according to claim 1, wherein the quercetin and astragalin are contained in an amount of 0.001 to 10% by weight, respectively, based on the total weight of the composition. The composition for external application of skin according to claim 1, wherein the composition is for anti-inflammatory. According to claim 1, wherein the composition is an external composition for skin, characterized in that for reducing the itch. Oral composition for improving atopic dermatitis containing quercetin and astragalin. The oral composition of claim 5, wherein the quercetin and astragalin are contained in an amount of 0.001 to 10% by weight, respectively, based on the total weight of the composition. 6. The oral composition of claim 5, wherein the composition is for anti-inflammatory. 6. The oral composition of claim 5, wherein the composition is for reducing itch.

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