KR101209208B1 - Composition comprising sesquiterpene lactone compounds for prevention or treatment of allergy - Google Patents

Abstract

The present invention relates to a composition for preventing or treating allergy, comprising a sesquiterpene lactone compound.

Description

Composition comprising sesquiterpene lactone compounds for prevention or treatment of allergy

The present invention is derived from research conducted as part of the General Researcher Support Program of the Ministry of Education, Science and Technology / Korea Research Foundation.

[Project ID: 500-20090279, Assignment Name: Study on mechanism of action of atopy inhibitors involved in the regulation of interleukin-induced signaling molecules]

The present invention relates to a composition for preventing or treating allergy, comprising a sesquiterpene lactone compound.

The incidence of irritable or inflammatory immune diseases increases with the change of living environment due to industrialization and westernization, and it is a serious social problem due to the contraction of social or economic activities. Inflammatory / sensitizing immune diseases are progressive immune responses that appear to be compromised in the balance of immune tolerance and immune responses, as are the main causes of many other immune diseases. Such inflammatory / irritable immune disease refers to a case in which a defense mechanism acting to protect a living body adversely affects a living body and causes a disorder, and is widely known as an allergy or allergy.

Allergic reactions are initiated by the influx of antigens into the body. The introduced antigen is recognized by antigen presenting cells, whereby T helper cells (Th) differentiate into Th helper 1 cells and Th helper 2 cells. Cells differentiated by antigens take the form of Th 2 cells. This differentiation results in an imbalance between the Th1 and Th2 responses, leading to allergic diseases. To balance Th immune responses, Th1 forms of cytokines such as interferon-gamma are also used as inhibitors of allergic diseases (Chai, SK, Altman, GM, Yazdanbakhsh, M., Tsuji, J., Godat, L.). & Takaro, TK (2005). Production of interleukin 10 and transforming growth factor beta in concomitant allergy and autoimmunity. Ann Allergy Asthma Immunol 94 (2): 279-285 ).

Allergies can be divided into five categories: first form: hypersensitivity reaction (anaphylactic shock), second form: antibody mediated cytotoxic reaction (autoimmune hemolytic anemia), third form: immune complex form (glomerulonephritis), Fourth form: cell-immune form (tuberculin response), and fifth form: stimulus response (Grave's disease).

Among them, the first type of allergic reaction is a reaction induced by the interaction between the antigen and the specific IgE to the antigen, and occurs in various allergic diseases, in particular, mast cells are involved in allergic and inflammatory diseases. Mast cells are present in the mucosal surface vascular tissues of the intestine and skin, secrete inflammatory mediators and cytokines, and affect cell tracing (Beaven, MA & Metzger, H. (1993). Signal transduction by Fc receptors: the Fc epsilon RI case.Imunmunol Today 14 (5): 222-226; Bochner, BS & Schleimer, RP (2001). Mast cells, basophils, and eosinophils: distinct but overlapping pathways for recruitment. Immunol Rev 179: 5-15). Interleukin-4 (IL-4) is a cytokine that differentiates Th0 cells into Th2 cells. IL-4 induces the conversion of B cells to IgE and is involved in allergic reactions as it is overproduced, infiltrating inflammatory cells and causing late stage allergic reactions (Metcalfe, DD, Baram, D. & Mekori, YA (1997)). Mast cells.Physiol Rev 77 (4): 1033-1079).

IL-5 is a another major pro-inflammatory factors (Hamelmann, E., Wahn, U. & Gelfand, EW (1999). Role of the Th2 cytokines in the development of allergen-induced airway inflammation and hyperresponsiveness. Int Arch Allergy Immunol 118 (2-4): 90-94), when mast cells are stimulated by the antigen-IgE complex, they are released from Th2 cells and increase eosinophil inflammation and mucus.

Allergic diseases continue to increase with changes in living environment and increase of pollutants due to industrialization, becoming the most common chronic diseases. However, despite the active research to date, the lack of proper treatment and preventive methods cause deterioration of quality of life and economic loss.

Accordingly, the present inventors have conducted studies on the treatment of allergic diseases, and sesquiterpene lactones induce the secretion of allergen-derived particulates such as β-hexoaminidase and IL-4 in RBL-2H3. The present invention was found to have the effect of inhibiting and inhibiting the activity of inflammatory cytokines such as IL-5 in Y16 cells.

It is an object of the present invention to provide a composition for the prevention or treatment of allergy, comprising a sesquiterpene lactone compound.

It is another object of the present invention to provide a process for preparing sesquiterpene lactone compounds from natural materials.

The present invention provides a composition for the prevention or treatment of allergy comprising a sesquiterpene lactone compound.

In one embodiment of the invention, the sesquiterpene lactone compound may be selected from the group consisting of Costunolide, Magnolialide, and Zaluzanin D having the structure shown below But not limited to:

Costunolide

Magnoliaid

Zaluzanin D

As used herein, the "sesquiterpene compound" is a kind of terpene-based compound, and is widely distributed in various plants. In particular, sesquiterpene lactone having a lactone structure is one of a group of plant natural compounds in which various physiological actions have been reported. This compound group is known to have antitumor activity, cytotoxicity, antibacterial action, and insecticidal action, and pharmacological activity such as gastrointestinal protective function and analgesic action has also been reported.

As used herein, the term "allergic disease" or "allergic" refers to various diseases, diseases or abnormalities caused by hypersensitivity to a specific substance of the human body, ie, excessive reaction of the immune system to a substance from outside. In particular, it refers to hypersensitivity in which an inflammatory mediator, such as histamine, is released by a substance from outside, causing disease.

In one embodiment of the invention, the allergic disease or allergy may be an allergic disease (type 1 allergic reaction) mediated by IgE.

In one embodiment of the invention, the allergy may be selected from the group consisting of bronchial asthma, allergic rhinitis, atopic dermatitis, allergic conjunctivitis, allergic otitis media, urticaria and anaphylatic shock.

In one embodiment of the invention, the sesquiterpene lactone compound may be derived from an extract of the leaves of Laurus nobilis L ..

In one embodiment of the invention, the sesquiterpene lactone compound is

Cutting the laurel leaf dry medicinal herbs and then warming to 40 to 50 ℃ in water-soluble alcohol, and then filtering to obtain an alcohol extract,

Concentrating the alcohol extract under reduced pressure, extracting by adding hexane and chloroform, respectively, to obtain a hexane layer fraction and a chloroform layer fraction,

The hexane layer fractions and the chloroform layer fractions may be obtained by a method comprising concentration under reduced pressure, respectively, and separating by HPLC.

In one embodiment of the invention, the water soluble alcohol used for the extraction of the bay leaf may be methanol or ethanol.

In addition, the compositions of the present invention may be prepared as pharmaceutical compositions or food compositions.

When the composition of the present invention is manufactured from a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are conventionally used in the formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, silicic acid Calcium, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. Do not. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and additives are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical compositions of the present invention may be administered by oral or parenteral administration, for example, by intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, or topical administration.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . The dosage of the pharmaceutical composition of the present invention may preferably be 0.01-100 mg / kg body weight per day.

The pharmaceutical compositions of the present invention are prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container. The formulation of the pharmaceutical composition of the present invention is suitable for pharmaceutical preparations, including powders, granules, tablets, capsules, suspensions, oral formulations such as emulsions, syrups, aerosols, external preparations such as ointments, creams, suppositories, and sterile injectable solutions. It may be a formulation, and may further include a dispersant or stabilizer.

When the composition of the present invention is made of a food composition, in addition to the active ingredient of the present invention described above, it includes ingredients that are conventionally added at the time of food production, for example, proteins, carbohydrates, fats, nutrients, seasonings and flavors It may include the agent.

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

The composition comprising a sesquiterpene lactone compound according to the present invention can be usefully used for the prevention or treatment of allergic diseases by inhibiting the secretion of allergen-causing fine particles and the expression of inflammatory cytokines.

1 shows cytotoxic effects of sesquiterpene lactones, costunolides (1a), magnolialides (1b), and zaluzanin D (1c) on RBL-2H3 cells according to one embodiment of the invention. .
FIG. 2 shows the inhibitory effects of costunolide (2a), magnolide (2b), and zaluzanin D (3c) on beta-hexosaminidase release in RBL-2H3 cells. .
FIG. 3 shows the effects of costunolide, magnolide and zaluzanin D on IL-4 mRNA expression in RBL-2H3. 3a, 3c and 3d are RT-PCR results, 3b. 3e and 3f are graphs quantifying the RT-PCR results.
Figure 4 shows the effect of ketotifen, a positive control substance on IL-4 mRNA expression in RBL-2H3 cells RT-PCR results (4a) and quantified graph (4b).
FIG. 5 shows the inhibitory effects of costunolid (5a), magnolide (5b), and zaluzanin D (5c) on IL-5 dependent Y16 cell proliferation.

Example  One. Sesquiterpenes  Preparation of Lactone Compounds

The sesquiterpene lactone compound, costunolide, magnolide, and zaluzanin D were isolated from the laurel leaves.

First, 20 L of 100% ethanol was added six times to 10 kg of dried laurel leaves, which were dried and dried, and extracted for 48 hours at 55 ° C. to extract 2.4 kg of an ethanol layer. The 100% ethanol extract layer obtained above was concentrated under reduced pressure, and 20L of chloroform was added six times to fractionate the chloroform layer to obtain 282.79 g of a chloroform layer. 20 L of hexane was added to the methanol extract, and the mixture was repeatedly extracted six times. The mixture was separated in a separatory funnel, and the hexane layer and the ethanol layer were separated. The hexane layer was obtained, and the hexane layer was concentrated under reduced pressure to obtain a hexane layer fraction. .

The hexane layer fraction of the laurel leaf obtained above was separated by chromatography on a silica gel column (YMC-Pack SIL), and the mixed solvent (20% ethyl acetate / hexane) of hexane and ethyl acetate was monitored by TLC. 10 fractions were obtained. Fraction 5 was then separated by HPLC using a normal phase silica column (YMC-Pack SIL), eluting with a mixture of ethyl acetate and hexane (15% ethyl acetate / hexanes), for a total of 13 fractions. Was obtained, and pure costunolide was isolated from the sixth fraction. Isolated compounds were identified by comparison with published data via ¹ H-NMR, ¹³ C-NMR, COZY, and HSQC (Juan F Sanz, Gloria Castellane and J. Alberto Marco, Sesquiterpene Lactones from ARTEMISIA HERBA alba ).

The chloroform layer fractions of the laurel leaves obtained above were separated by chromatography on a silica gel column (YMC-Pack SIL) and the fractions were monitored by TLC, with a mixed solvent of hexane and ethyl acetate, acetonitrile and methanol gradient (100% hexane , 10% Ethyl Acetate / 90% Hexane, 20% Ethyl Acetate / 80% Hexane, 30% Ethyl Acetate / 70% Hexane, 40% Ethyl Acetate / 60% Hexane, 50% Ethyl Acetate / 50% Hexane, 60% Ethyl Acetate Eluted with (40% hexane, 70% ethyl acetate / 30% hexane, 100% ethyl acetate, 100% acetonitrile, 100% methanol).

Thereafter, fraction 6 was separated by HPLC using a normal silica column and eluted with a mixture of ethyl acetate and hexane (25% ethyl acetate / hexane) to obtain a total of seven fractions. Alid was obtained.

In addition, fraction 4 was separated by HPLC using a normal silica column and eluted with a mixture of ethyl acetate and hexane (12% ethyl acetate / hexane) to obtain pure zaluzanin D in fraction 6. Isolated compounds were identified by comparison with published data via ¹ H-NMR, ¹³ C-NMR, COZY, and HSQC (Sevil Oksuz and Gulacti Topin, Eudesmanolide and other constituents from INULA). GRAVEOLENS ).

Example  2. Sesquiterpenes  Anti-allergic action of lactone compounds

In this example, the anti-allergic effect of the sesquiterpene lactone compound obtained in Example 1 was confirmed.

All experimental results described below are expressed as mean ± standard deviation, and statistical significance was determined by the t-test (Sigma Plot 10.0. Software, SPSS, USA) and considered statistically significant for p <0.05. .

2-1. RBL -2 H3  Cell Culture and Cytotoxicity

RBL-2H3 cells are very similar to basophilic and mast cells, express rapidly IL-4, and are the most convenient and widely used cells in the study of particulate secretion of mast cells. On the surface of these cells, many receptors FcεRI have strong binding to IgE, and this IgE-FcεRI binding is induced by a large amount of antigen. From the cells by the reaction of histamine (Barsumian, EL, Isersky, C. , Petrino, MG & Siraganian, RP (1981) IgE-induced histamine release from rat basophilic leukemia cell lines:.. Isolation of releasing and nonreleasing clones Eur J Immunol 11 (4): 317-323) , serotonin (Taurog, JD, Fewtrell, C. & Becker, EL (1979) IgE mediated triggering of rat basophil leukemia cells:.. lack of evidence for serine esterase activation J Immunol 122 ( 6): 2150-2153), and beta-amino hexose kinase I (Schwartz, LB, Austen, KF & Wasserman, SI (1979) immunologic release of beta-hexosaminidase and beta-glucuronidase from purified rat serosal mast cells J Immunol.. Allergic chemicals such as 123 (4): 1445-1450) are secreted. These substances play a major role in causing major allergic symptoms such as vasodilation, mucus secretion and bronchodilation.

To study anti-allergic effects, RBL-2H3 cells (Korea Cell Line Bank, Korea) contain 8% fetal bovine serum (FBS) (Gibco-BRL, USA) and antibiotics (Sigma-Aldrich, USA) Incubated in DMEM (Dulbecco's Modified Eagle's Medium) (Sigma-Aldrich, USA) medium at constant temperature of 37 ℃, 5% CO ₂ .

The cytotoxicity of the sesquiterpene lactone compounds was tested against RBL-2H3 cells. Cell viability was confirmed using the MTT assay. RBL-2H3 cells ( ⁵ × 10 ⁵ cells / ml) were inoculated in 100 μl / well of culture medium in 96-well plates and cultured under the conditions described above. After 24 hours of incubation, the cells were reacted with various concentrations of sesquiterpene lactone compounds for 4 hours, followed by MTT (3- (4,5-dimethylthiazol-2yl) -2,5-diphenyl-tetrazolium bromide ) (250 μg / ml) and incubated at 37 ° C. for 3 hours. Then, sodium dodecyl sulfate (SDS) solution (50% DMF and 20% SDS in water) was added to 100 µl of each well to dissolve formazan crystals. Cell viability was measured by absorbance at 595 nm, and compared with the control group not treated with the sesquiterpene lactone compound at 100% survival.

1 shows the effect of sesquiterpene lactone compounds on the viability of RBL-2H3 cells. RBL-2H3 cells showed a 95% survival rate compared to the control at sesquiterpene lactone concentrations of 0.32 μM to 40 μM.

2-2. RBL -2 H3  Beta-in Cells Hexosaminidase  Secretion inhibition

RBL-2H3 cells ( ⁵ × 10 ⁵ cells / ml) were incubated in 100 μl medium at a concentration of ⁵ × 10 ⁴ cells / well. In culture, cells were sensitized with anti-DNP-IgE (Sigma-Aldrich, St. Louse, Mo, USA) at a final concentration of 1 μg / ml. The cells obtained after the culture were washed twice with HEPES buffer to remove the remaining IgE without binding to the cells. The cells were then separated with a sesquiterpene lactone compound and 45 μl of a releasing mixture (116.9 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO ₄ .7H ₂ O, 5.6 mM glucose, 25 mM HEPES, 2.0 mM CaCl) per well. ₂ and 1.0 mg / ml BSA at pH 7.7) for 20 minutes. Subsequently, 5 µl of DNP-BSA at a concentration of 4 µg / ml dissolved in PBS was added to the cells, followed by reaction for 1 hour. After the reaction, only the supernatant was separated from the plate and transferred to a new 96-well plate, and 50 μl of substrate solution (50 mM citric acid buffer, 5 mM 4-nitrophenyl N-acetyl-β-D-glucosamide in pH 4.5) was added. Then incubated at 37 ° C. for 90 minutes. The reaction was terminated by the addition of 50 μl of stopping buffer (0.5 M Na ₂ CO ₃ / NaHCO ₃ , pH 10.0). Absorbance was measured at 405 nm in microplate reader.

Negative controls were not treated with test samples and positive controls were treated with ketotifen instead of sesquiterpene lactone compounds. Ketotifen is frequently used with RBL-2H3 cells for immune response and allergy studies as an inhibitor of IgE-mediated degranulation. Ketotifen is known to selectively regulate histamine release in tissue binding mast cells and to inhibit 48 / 80-stimulated histamine release.

Inhibition of release of beta-hexosaminidase by the test sample (%) was calculated by the following formula.

Inhibition (%) = [1- (T-C) / (I-C)] x100

Control (C): Cell (+), DNP-IgE (-), Test Sample (-)

Test (T): Cell (+), DNP-IgE (+), Test Sample (+)

Inducer (I): Cell (+), DNP-IgE (+), Test Sample (-)

In order to measure allergen-induced particulate secretion in RBL-2H3 cells, the immune response was induced with Dg-BSA after sensitization with IgE. The amount secreted by the microparticles was measured by beta-hexosaminidase secretion. Treatment of the sesquiterpene lactone compound resulted in a concentration-dependent decrease in the amount of beta-hexosaminidase secretion in RBL-2H3 cells. Table 1 and FIG. 2 below show the results.

Substance / concentration 0.32 μM 1.6 [mu] M 8 μM 40 μM Costunolide 2.4 ± 1.9% 4.9 ± 0.3% 13.7 ± 1.9% 36.5 ± 3., 9% Magololyid 3.4 ± 3.4% 7.0 ± 1.8% 15.5 ± 0.8% 57.9 ± 1.1% Zaluzanin D 3.4 ± 2.9% 4.4 ± 2.9% 10.1 ± 2.2% 44.8 ± 5.3%

Antigen-IgE binds to FcεRI with a strong binding to IgE and induces the secretion of fine particles of mast cells, namely allergic mediators such as histamine, serotonin and beta-hexosamidinase (Siraganian, RP (2003)). Mast cell signal transduction from the high- affinity IgE receptor. Curr Opin Immunol 15 (6): 639-646 ). Through the above results, it was confirmed that the sesquiterpene lactone compound including costunolid, magnolide, and zaluzanin D extracted from the laurel leaves inhibits beta-hexosaminidase secretion. In particular, magnolialide showed a better inhibitory effect than ketotifen, a positive control.

2-3. Using polymerase chain reaction IL -4 mRNA  Expression level measurement

Under Th2 immune responses, mast cells and T cells produce Th2 cytokines IL-4, IL-5 and IL-13. IL-4 maintains cell activation and further enhances Th2 immune response. In particular, IL-4 induces the growth of basophils and mast cells and acts as a variable factor for lymphocytes (Ricci, M., Matucci, A. & Rossi, O. (1997b) .Source of IL-4 able to induce the development of TH2-like cells.Clin Exp Allergy 27 (5): 488-500 ). IL-4 and IL-13 promote the conversion of B cells to IgE through isotype modification. IL-4 increases Th2 cytokine expression and plays an important role in the activation of mast cells through B cells and allergic diseases. In this example, the inhibitory effect of the sesquiterpene lactone compound on the IL-4 cytokine production of RBL-2H3 cells was evaluated.

RBL-2H3 cells ( ⁵ × 10 ⁵ cells / mL) were incubated in 1 ml medium at ⁵ × 10 ⁴ cells / well concentration in 12-well plates. While culturing the cells, they were sensitized at 37 ° C. for 24 hours with anti-DNP-IgE at a concentration of 1 μg / ml. Cultured cells were washed twice with HEPES buffer to remove unbound IgE. The washed cells were washed with 70 μl of sesquiterpene lactone compound and 630 μl of release mixture per well (116.9 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO ₄ .7H ₂ O, 5.6 mM glucose, 25 mM HEPES, 2.0 mM CaCl ₂ and 1.0 mg / ml BSA, pH 7.7) at 37 ° C. for 3 hours.

Thereafter, 70 μl of 4 μg / ml DNP-BSA per well was added to induce an immune response at 37 ° C. for 1 hour. After antigen stimulation, total RNA was extracted using easy-BLUE ™ Total RNA Extraction Kit (Intron Biotechnology, Korea). CDNA was synthesized using 1 ㎍ of RNA and ONE-STEP RT-PCR premix kit (Intron Biotechnology, Korea). The following primers were used for cDNA synthesis:

IL-4:

Forward primer 5'-ACCTTGCTGTCACCCTGTTC-3 '(SEQ ID NO: 1)

Reverse primer 5'-TTGTGAGCGTGGACTCATTC-3 '(SEQ ID NO: 2)

β-actin:

Forward Primer 5'-GACCCAGATCATGTTTGAGA-3 '

Reverse primer 5'-GCTTGCTGATCCACATCTGC-3 '

The PCR reaction was performed using GeneAmp PCR System 9700 (Applied Biosystems) in 35 cycles consisting of 45 seconds denaturation at 94 ° C., 45 seconds annealing at 58 ° C. and 45 seconds elongation at 72 ° C. for amplification of IL-4. , 25 cycles consisting of 45 seconds denaturation at 94 ° C., 45 seconds annealing at 55 ° C. and 45 seconds elongation at 72 ° C. were performed for amplification of β-actin. PCR reaction products were run by electrophoresis on 1.5% (w / v) agarose gel and confirmed by staining with SYBR Safe DNA gel stain (Invitrogen, CA, USA). MRAN expression was measured using an image analyzer (LAS 1000 plus, Fuji photo film, JAPAN) and Multi Gauge software (Fuji photo film, JAPAN) program, and was determined in comparison to β-actin as a control. The size of the band was confirmed using a molecular size marker (100 bp DNA Ladder Marker, Takara).

Figure 3 shows the effect of sesquiterpene lactone compound on the amount of IL-4 mRNA expression. Costunolide inhibited IL-4 mRNA expression in RBL-2H3 cells in a concentration-dependent manner (IC ₅₀ = 34 μM) (FIGS. 3A and 3B). Magnolide also showed excellent efficacy in inhibiting IL-4 production (IC ₅₀ = 22 μM) (FIGS. 3C and 3D), and also, zaluzanin D also concentration-dependently reduced IL-4 expression (IC ₅₀ = 28 μM). ) (FIGS. 3E and 3F).

4 shows the effect of ketotifen, a positive control substance on IL-4 mRNA expression in RBL-2H3 cells, RT-PCR results (4a), and a quantified graph (4b). Positive controls were treated with ketotifen, which is known to reduce IL-4 expression in RBL-2H3 cells as an antiallergic agent (IC ₅₀ = 24 μM) (FIG. 4A). Sesquiterpene lactone compounds have been found to have similar or better IL-4 expression inhibition efficacy than ketotifen.

2-4. IL -5 dependencies Y16  Inhibition of cell proliferation

Y16 cells are B cells in their initial state and proliferate depending on the presence of IL-5. In this example, the proliferation of IL-5 dependent Y16 cells was measured to investigate the effects of sesquiterpene lactone compounds on IL-5 activity.

Y16 cells (Professor K. Takatsu, University of Tokyo) were prepared in RPMI (10.4 mg / ml RPMI-1640, 24 mM NaHCO ₃ , 100X antibiotic (Sigma), 50 μΜ 2-mercaptoethanol) at a final concentration of 10 U / ml mIL-5 Incubated at 37 ° C., 5% CO ₂ . After incubation, the cells were collected by centrifugation at 1000 rpm for 10 minutes, then suspended in culture, dispensed in 100 μl of 5 × 10 ⁴ cells / well into 96-well microplates, and the final concentrations of 10 U / ml of mIL-5 and 10 A sesquiterpene lactone compound in% DMSO was added. The control group added only 10% DMSO instead of sesquiterpene lactone, and did not treat mIL-5, and the Inducer group added RPMI of 8% FBS after adding 10% DMSO and mIL-5 instead of sesquiterpene lactone. Incubated in the medium. After 48 hours of incubation at 37 ° C. and 5% CO ₂ , the cells were reacted with MTT (3- (4,5-dimethylthiazol-2yl) -2,5-diphenyltetrazolium bromide) (250 μg / ml) for 3 hours. After the reaction, 100 μl of sodium dodecyl sulfate (SDS) (50% DMF and 20% SDS in water) was added per well and reacted for 24 hours. Viability of Y16 cells was measured at ₅₄₀ nm ( A ₅₄₀ ) using a microplate reader (Molecular Device, USA).

The inhibitory effect on IL-5 was calculated according to the following equation:

Inhibition (%) = [1- (Sample A ₅₄₀ -Control A ₅₄₀ ) / (Inducer A ₅₄₀ -Control A ₅₄₀ )] x100

IL-5 cytokines are known to play an important role in allergic diseases. Sesquiterpene lactone compounds inhibited mIL-5 activity in Y16 cells. In the group treated with the sesquiterpene lactone compound, the amount of IL-5 dependent Y16 cells decreased compared to the groups in which the immune response was induced by the inducer. All experiments were performed at concentrations of 0.16 μM, 0.8 μM, 4 μM and 20 μM of sesquiterpene lactone compounds. The table below shows the reduction of IL-5 dependent Y16 cells by sesquiterpene lactone compounds compared to the control.

Substance / concentration 0.16 μM 0.8 μM 4 μM 20 μM Costunolide 6.3 ± 0.7% 10.4 ± 2.9% 27.5 ± 5.9% 34.0 ± 11.2% Magnoliaid 9.4 ± 1.0% 14.2 ± 6.8% 29.6 ± 7.2% 56.2 ± 0.7% Zaluzanin D 16.5 ± 5.1% 20.7 ± 6.7% 43.2 ± 2.5% 71.3 ± 8.3%

In particular, Zaluzanin D showed a high inhibition rate of 71.3% compared to the group in which the immune response was induced by the inducer. These results reflect that sesquiterpene lactone compounds have IL-5 antagonism and inhibitory efficacy.

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Claims (3)

A composition for preventing or treating allergy, comprising a sesquiterpene lactone compound selected from the group consisting of costunolide, magnolialide, and zaluzanin D as an active ingredient. The composition of claim 1, wherein the allergy is selected from the group consisting of bronchial asthma, allergic rhinitis, atopic dermatitis, allergic conjunctivitis, allergic otitis media, urticaria, and anaphylatic shock. delete

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