KR20150112624A - Composition for preventing or treating asthma comprising the extracts of Distylium racemosum - Google Patents

Abstract

The present invention relates to a composition for preventing or treating asthma containing an extract of Distylium racemosum, as an active ingredient. According to the present invention, the extract of Distylium racemosum inhibits activities of both an S-nitrosoglutathione (GSNO) reductase and a phosphodiesterase-4A1 (PDE4A1), thereby being able to be used as an effective composition for preventing or treating asthma.

Description

TECHNICAL FIELD The present invention relates to a composition for prevention or treatment of asthma,

The present invention relates to a composition for preventing or treating asthma, and more particularly to wood jorok (Distylium racemosum ) extract as an active ingredient.

Asthma is an inflammatory allergic disease of the airways, the main symptom of which is coughing and dyspnea with wheezing. Pneumonia with chronic inflammation is exposed to various risk factors, leading to muscle contraction, increased mucus secretion, and increased erythema / edema, which results in airflow limitation and airway obstruction. The etiology of asthma is diverse and is often divided into extrinsic asthma, which is caused by exposure to external factors (such as house dust, mites, pollen, fungi, etc.), and endogenous asthma caused by changes in airway infections and humidity. When the airway is exposed to the above various pathologies, macrophages, Th2 cells, eosinophils, etc. involved in airway inflammation are activated and various inflammatory mediators such as histamine and leukotrienes are secreted to induce airway hyperresponsiveness. Since asthma involves various inflammatory cells and a variety of inflammatory mediators released therefrom, it is difficult to expect clinically sufficient pharmacological effects by treatment of specific targets due to the characteristics of asthma diseases.

Asthma medications that have been developed to date can be classified as drugs that alleviate symptoms of airway obstruction (such as bronchodilators) and medications that suppress airway inflammation.

Drugs used as bronchodilators include beta 2 agonists, anticholinergics, and theophylline, but various side effects have been reported. The beta 2 agonist binds to the beta 2 receptor of the bronchial smooth muscle to activate adenylate cyclase, thereby increasing the amount of cAMP, thereby causing myosin light chain kinase (MLCK), a cAMP dependent protein, to be phosphorylated in the bronchial smooth muscle, As the smooth muscle relaxes, bronchial dilatation can occur. The theophylline agent inhibits airway infiltration of T cells and eosinophils as a non-specific phosphodiesterase inhibitor and induces eosinophil cell death.

Drugs that inhibit airway inflammation include steroids, anti-leukotrienes, and cromolynes, which inhibit inflammatory cell infiltration, decrease blood vessel permeability, inhibit airway secretion, inhibit airway hyperresponsiveness, inhibit cytokine production . These drugs, however, are not considered to be the primary remedy for asthma, either by alleviating each symptom of asthma or simply being used as an interim treatment in emergencies.

Thus, along with the study of new points of action for the fundamental treatment of asthma, it is urgent to develop drugs that can control the point of action.

Recently, PDE4 (phosphodiesterase 4) inhibitors and GSNO reductase inhibitors have been actively studied as a new point of action for the fundamental treatment of asthma.

PDE4 is an enzyme which is expressed in all major inflammatory cells and inactivates cyclic 3 ', 5'-adenosine monophosphate (cAMP). When the activity of PDE4 is inhibited, the amount of intracellular cAMP can be increased to induce anti-inflammatory action due to relaxation of the smooth muscle in the airway and deterioration of immune cell function. Recently, Daxas (Roflumilast), which has been attracting attention as a representative PDE4 inhibitor, has been approved as an agent for the treatment of chronic obstructive pulmonary disease with symptoms similar to those of asthma. In addition to the above Daxas, several PDE4 inhibitors have been developed as asthma treatment drugs, but their development is stopped or their licenses are delayed due to side effects such as vomiting (Higgs, G. 2010, Is PDE4 too difficult a drug target, Curr Opin Investig Drugs, 11 (5), 495-8.).

GSNO reductase is a class III alcohol dehydrogenase (ADH) that regulates the metabolism of GSNO (S-nitrosoglutathione), an endogenous bronchodilator. GSNO is produced by the reaction of glutathione and nitric oxide in cells and serves as a reservoir of stable nitric oxide. That is, although the nitric oxide itself has a short half-life to exhibit its activity, it has been reported that GSNO can maintain homeostasis of nitrogen oxide by acting as a reservoir of nitric oxide. The amount of GSNO reductase in the airway increased as a result of the use of ovalbumin, which is frequently used as an asthmatic inducer in experimental animals, as an antigen. As a result, the amount of GSNO decreased. Thus, the reduction of GSNO has been shown to be directly related to the hypersensitivity of airway. On the other hand, the amount of GSNO was increased in experimental animals in which GSNO did not express lidocaine, and as a result, no airway hyperreactivity was caused by the albumin antigen. These results indicate that GSNO metabolized by GSNO reductase is associated with airway hyperresponsiveness, suggesting that a drug that inhibits the activity of GSNO reductase can be developed as a therapeutic agent for asthma. In fact, N30 Pharmaceuticals has been conducting clinical trials of the N6022 compound, an inhibitor of GSNO reductase (Green, LS et al, 2012, Mechanism of inhibition for N6022, a first-in-class drug targeting S-nitrosoglutathione reductase, 51 (10), 2157-2168).

Thus, the development of asthma treatment agents capable of inhibiting the enzymatic activity of PDE4 and GSNO reductase has been actively carried out. The present invention has been completed by confirming the PDE4 and GSNO reductase activity inhibitory activities of P. japonica extract will be.

It is an object of the present invention to provide a composition for preventing or treating asthma having inhibitory activity of enzyme activity of PDE4 and GSNO reductase using an extract of Aspergillus oryzae as an active ingredient.

Other and further objects of the invention will be set forth hereinafter.

The present invention relates to a composition for preventing or treating asthma comprising astragalus extract as an active ingredient, as confirmed in the following examples and experimental examples. The present inventors screened the extracts of the plant-derived plant extracts from Jeju area to inhibit the enzymatic activity of PDE4A1 and the enzyme-inhibiting activity of GSNO reductase, Respectively. The enzyme inhibitory activity and the IC50 value of fractions obtained by sequentially fractionating these extracts with n - hexane, ethyl acetate and butanol were measured. The present invention is based on the results of these experiments. When the activity of GSNO reductase and PDE4A1 enzyme is inhibited, the in vivo asthma reaction is inhibited. Accordingly, the composition of the present invention is provided as a composition for preventing and treating asthma .

As an active ingredient of the present invention, the distriium racemosum is also called a gourd tree or a mourning bag. It is a dicotyledonous plant belonging to Rosaceae Rosaceae, distributed in Korea (southern), Japan, and China. Leaves are oval or oval with 3 ~ 8cm in length, 1.5 ~ 3cm in width, with slanting leaves. There are no hairs on the both sides of the leaves, no hairs, and flat edges. The flowers are bloomed in April, blossomed and bloom on the axil, with the upper part bisexual and the lower part with male flowers. The fruit is capillary, ripened in September-October, and is 1 ~ 1.5cm long woody, with dense hairs on the surface, and the seed is split into two pieces.

The timber can be roots, stems, branches, leaves, flowers, seeds and / or fruits, preferably leaves and / or branches.

In the present specification, the term "extract" refers to any substance which is extracted regardless of the extraction method, such as water, lower alcohol having 1 to 4 carbon atoms such as methanol, ethanol and butanol, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, The crude extract obtained by using methane, N, N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), 1,3-butylene glycol, propylene glycol or a mixed solvent thereof and its crude extract are dissolved in the above- And a fraction obtained by fractionating at least one of them. A fraction obtained by suspending the crude extract in a specific solvent and then mixing and leaving with a solvent having a different polarity, a step of adsorbing the crude extract on a column packed with silica gel or the like, and then adding a hydrophobic solvent, a hydrophilic solvent, Means a fraction obtained by using a mixed solvent as a mobile phase. Also, the meaning of the above extract includes a concentrated liquid extract or a solid extract in which a part of the extraction solvent is removed by freeze drying, vacuum drying, hot air drying, spray drying and the like. Preferably, as a solvent for extraction, a fraction of a fraction obtained by fractionating water, hexane, water and ethyl acetate or water and butanol obtained by extracting a solid form of the extract obtained by using water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, Or an extract thereof is suspended in water and then fractionated successively with hexane, ethyl acetate and butanol. Here, the term "sequential fractionation" means that the fraction of the water layer is continuously used after fractionation and is fractionated into the solvent of the above-mentioned order.

As used herein, the term "active ingredient" alone means an ingredient which exhibits the desired activity or which can exhibit activity together with a carrier which itself is not active.

The effective ingredient of the composition of the present invention may be included in any amount (effective amount) depending on the purpose of use, formulation and formulation, so long as it can exhibit asthma prevention and treatment effect. And will be determined within the range of 0.0001% by weight to 99.9000% by weight, based on the total weight of the composition. Here, "effective amount" refers to the amount of active ingredient capable of inducing the preventive and therapeutic effect of asthma. Such effective amounts can be determined experimentally within the ordinary skill of those skilled in the art.

Meanwhile, the composition of the present invention can be used as a pharmaceutical composition for preventing or treating asthma.

The pharmaceutical composition of the present invention may be prepared by mixing an effective component of the Algerian root extract with an additive such as a pharmaceutically acceptable carrier, excipient, stabilizer, preservative, buffer, mating agent, flavoring agent, sweetener, Formulation is intended for oral administration. The additive refers to a substance that is further used to improve the stability, safety, and quality when formulating pharmaceuticals.

Examples of the solid composition for oral administration include tablets, pills, capsules, powders, granules and the like. In such a solid composition, the plant extract as an active ingredient is mixed with at least one inert diluent such as lactose, mannitol, glucose, hydroxypropylcellulose, microcrystalline cellulose, starch, polyvinylpyrrolidone, magnesium aluminometasilicate, Can be mixed. The solid composition may contain additives other than an inert diluent, for example, a lubricant such as magnesium stearate, a disintegrant such as calcium cellulose glycolate, a solubilizing agent such as glutamic acid or aspartic acid, according to a conventional method. The tablets or pills may be coated with a film made of a material such as sucrose, gelatin, hydroxypropylmethylcellulose phthalate or the like, if necessary, and may be coated with two or more layers as the case requires.

The liquid composition for oral administration may contain a generally used inert diluent such as purified water, ethanol, etc., including pharmaceutically acceptable emulsions, solutions, suspensions, syrups, elixirs and the like. The composition may contain, in addition to an inert diluent, a wetting agent, an adjuvant such as a suspending agent, a sweetening agent, a flavoring agent, a fragrance, an antiseptic, and the like.

Injections for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsifiers. Examples of aqueous solutions and suspensions include water for injection and physiological saline for injection. Examples of the non-aqueous solution / suspension include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol, polysorbate 80® and the like. Such a composition may again contain adjuvants such as preservatives, wetting agents, emulsifying agents, dispersing agents, stabilizing agents (for example, lactose), and solubilizing agents (for example, glutamic acid, aspartic acid). These can be sterilized by a conventional sterilization method such as filtration sterilization with a microfiltration membrane, heat sterilization such as high pressure steam sterilization, or sterilizing agent combination. Injections can be solution formulations or freeze-dried preparations which can be used by dissolving in use. Examples of excipients for freeze-drying include sugar alcohols and sugars such as mannitol, glucose and the like.

The pharmaceutical composition of the present invention is administered by an oral administration method or a parenteral administration method.

The pharmaceutical composition may be administered orally, and may be administered by a parenteral administration method, for example, by injection (subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection or the like), transdermal administration, Administration (transdermal administration, etc.), transpulmonary administration, etc. may be used.

The dosage of the active ingredient of the pharmaceutical composition of the present invention may be determined according to the severity of the disease and the age of the patient, but is generally in the range of 0.005 / / kg to 100 mg / kg, preferably 0.02 / / 5 mg / kg. However, since the dosage of the pharmaceutical composition of the present invention is determined in view of various related factors such as route of administration, age, sex, weight, and patient's severity of the patient, the dose is limited in any aspect to the scope of the present invention It should not be understood.

The composition of the present invention may also be used as a food composition for preventing or treating asthma.

When the composition of the present invention is identified as a food composition, the form of the food is not particularly limited as far as it is a beverage such as tea, juice, carbonated beverage, ionic drink, processed oil such as milk and request route, gum, rice cake, Food preparations such as foods, powders, tablets, capsules and the like.

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

Sweetening agents may be used in an amount that sweetens the food in a suitable manner, and may be natural or synthetic. Preferably, natural sweeteners are used. Examples of natural sweeteners include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose and maltose.

Flavors may be used to enhance taste or flavor, both natural and synthetic. Preferably, a natural one is used. When using natural ones, the purpose of nutritional fortification can be performed in addition to the flavor. Examples of natural flavoring agents include those obtained from apples, lemons, citrus fruits, grapes, strawberries, peaches, and the like, or those obtained from green tea leaves, Asiatica, Daegu, Cinnamon, Chrysanthemum leaves and Jasmine. Also, those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, banks and the like can be used. The natural flavoring agent may be a liquid concentrate or a solid form of extract. Synthetic flavors may be used depending on the case, and synthetic flavors such as esters, alcohols, aldehydes, terpenes and the like may be used.

Examples of the physiologically active substance include catechins such as catechin, epicatechin, gallocatechin and epigallocatechin, and vitamins such as retinol, ascorbic acid, tocopherol, calciferol, thiamine and riboflavin.

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

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

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

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

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

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

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

Terms that are not specifically defined in this specification are intended to have a Korean national meaning or a meaning commonly used in the art.

FIG. 1A shows a gene cloning scheme of GSNO reductase.
In FIG. 1B, the right row shows the result of SDS-PAGE after separating and purifying GSNO reductase recombinant protein. The left column shows a band of standard proteins of known molecular weight.
FIG. 1C is a graph showing the results of measurement of absorbance change at 340 nm for 200 seconds when NADH is oxidized to NAD +. FIG. 1, GSNO reductase, GSNO reductase, GSNO reductase, and GSNO reductase, respectively. In line 2, GSNO reductase and substrate responses were inhibited by N6022 compound (GSNO reductase inhibitor) And the absorbance at 340 nm decreases with time as the NADH concentration decreases.
FIG. 2 is a graph showing the results of measuring the inhibitory effect of GSNO reductase on the extracts of 100 kinds of onshore plants native to Jeju Island. On the X-axis of the graph, 100 kinds of land plants are numbered from 1 to 100. Sample No. 71 is an extract of Alaska pollack.
FIG. 3A is a graph showing the inhibitory effect of GSNO reductase on the activity of fractions of Alaska pollack extract and leaf extract. FIG.
FIG. 3B is a graph showing the inhibitory effect of GSNO reductase on the fractions of Alnus japonica extract and Alnus japonica extract.
FIG. 4 is a graph showing the results of measuring the inhibitory effect of PDE4A1 on the extracts of 100 kinds of onshore plants native to Jeju Island. On the X-axis of the graph, 100 kinds of land plants are numbered from 1 to 100. Sample No. 71 is an extract of Alaska pollack.
FIG. 5A is a graph showing the inhibitory effect of PDE4A1 on the fractions of Allium cepa L. and Leaf extract.
FIG. 5B is a graph showing the inhibitory effect of PDE4A1 on the fractions of Allium cepa L. and Leaf extract.

Hereinafter, the present invention will be described with reference to Examples and Experimental Examples. However, the scope of the present invention is not limited to these examples and experimental examples.

< Example > Sample Preparation

<Example 1> jorok tree extract

<1-1> Prepare a timber sample

The leaves, stems and branches were used as samples. Trees were washed with water and dried in a hot air drier at 40 ° C for 3 days. The dried samples were finely pulverized using a pulverizer and used as extraction samples.

<1-2> Production of Alaska pollack extract

The samples used for the extraction are the leaves and branches of Alaska pollack. A 70% ethanol solvent was used to separate the water - soluble components from the leaves and branches. The extracted sample was added to a 70% ethanol solvent at a volume ratio of 1:20, and then the mixture was extracted with stirring for 24 hours. After extraction is complete, the ADVANTEC Grade No. 131 The extract was filtered using qualitative filter paper. Then, the filtrate was concentrated by a reduced pressure concentrator.

The extraction yield was 33.20% based on P. vivax. 20 g of the obtained leaf extract was suspended in water and then sequentially extracted with n-hexane, ethyl acetate and butanol. The fractions were lyophilized to obtain 0.5 g (2.5%) of n-hexane fraction and 4.1 g (20.5% 7.0 g (35%) of the butanol fraction and 9.0 g (45.0%) of the water fraction were obtained. The extraction yield was 18.00% on the basis of the tree branches. 20 g of the resulting eggplant extract was suspended in water and then sequentially extracted with n-hexane, ethyl acetate and butanol. The fractions were lyophilized to obtain 0.7 g (3.5%) of n-hexane fraction and 3.0 g (15.0% 7.0 g (35.0%) of the butanol fraction and 8.1 g (40.5%) of the water fraction were obtained. The obtained concentrate was prepared as a powder using a freeze dryer and stored in a freezer at -20 ° C until used in subsequent experiments.

&Lt; Example 2 > GSNO Reductase

<2-1> GSNO Gene cloning of reductase

RNA was extracted from neurospheres (Invitrogen) from cerebral cortex isolated from SD rat embryos on day 18 of pregnancy. CDNA was synthesized by reacting with a reverse transcriptase (Superscript reverse transcriptase, Invitrogen) and an oligo-d (T) 20 primer at 65 ° C for 5 minutes, 50 ° C for 50 minutes and 85 ° C for 5 minutes. Then, the gene was amplified from the cDNA using Platinum blue PCR superMix (Invitrogen). The base sequences of the corresponding primers are shown in Table 1.

GSNO reductase primer sequence Forward primer sequence 5'-GGATCCGCGAACCAGGTGATCAGG-3 ' Reverse primer sequence 5'-CGGCCGCGAGTGCAGGATGGGCAGTACT-3 '

PCR was carried out by repeating 35 cycles of denaturation at 94 DEG C for 5 minutes, denaturation at 94 DEG C for 30 seconds, binding at 55 DEG C for 30 seconds, and 72 DEG C for 1 minute and 30 seconds. The amplified cDNA was separated from 1% agarose gel, stained with ethidium bromide, and an inserted DNA fragment of 1,150 bp was prepared using an agarose gel separation kit (GeneAll).

The inserted cDNA and the T easy vector (promega) were placed in an EP tube and T4 DNA ligase (enzynomics) was added, followed by a binding reaction at 18 ° C for 6 hours. The reaction mixture thus obtained was added to competent cells and allowed to react on ice for 30 minutes, followed by heat shock reaction at 42 ° C for 1 minute. Subsequently, SOB medium was added thereto, Min. &Lt; / RTI &gt; Meanwhile, X-gal: IPTG was spread and dried on an LB agarose plate containing Ampicilin. The E. coli cultured for 40 minutes was centrifuged and submerged, spread on a previously prepared plate, and incubated in a 37 ° C incubator for 16 hours. Only white colonies among the colonies were selected for mass culture, and sequencing was performed to confirm the nucleotide sequence of GSNO reductase.

The pET-28a vector and the GSNO reductase / T easy vector were digested with restriction enzymes, respectively, to transfer the GSNO reductase thus identified from the T easy vector to the pET-28a vector tagged His at the N- After cleavage with BamH1 and Not1, the GSNO reductase-inserted fragment and the pET-28a vector were ligated in the same manner as described above (Fig. E. coli used for transformation was transfected with Rosetta2 (DE3) competent cells (Novagen) for 1 min at 42 ° C. The colonies were screened using kanamycin and the results were analyzed by sequencing. Respectively.

<2-2> GSNO Isolation and Purification of Reductase Recombinant Protein

A GSNO reductase plasmid cloned into a pET-28a vector having a histidine tag at its N-terminus was introduced into Rosetta2 (DE3) competent cells and transformed. To express GSNO reductase recombinant protein, the transformed cells were grown in LB medium for 2 hours (0.8 at OD 600 nm), and then incubated with 0.5 mM IPTG (isopropyl-1-thio-β-D-galactopyranoside) And the mixture was subjected to a transfection at 25 ° C for 6 hours. Proteins were separated using Ni-Sepharose high performance (GE healthcare) and imidazole (Sigma). Protein separation in the column was performed using 20 mM HEPES, pH 7.4 buffer and 250 mM NaCl, 10% glycerol, 0.1% Tween-20, 500 mM imidazole, 1% protein inhibitor. The separated proteins were put into dialysis buffer of 100 mM sodium phosphate pH 7.4, 100 mM KCl, 10% glycerol and stirred at 4 캜 for 24 hours to remove imidazole, and the buffer was exchanged with 100 mM sodium phosphate. The separated and purified proteins were electrophoresed on 10% SDS-PAGE and stained with Coomassie blue to be shown in FIG. 1b.

< Experimental Example > Anti-asthma  Verify Active

<Experimental Example 1> GSNO Activity measurement of reductase activity

In this experimental example, it was confirmed whether the activity of GSNO reductase could be inhibited by each extract of 100 kinds of land plants of Jeju Island.

GSNO reductase reduces GSNO and NADH, which is a reduced form of coenzyme NAD, is oxidized and converted to NAD +. NADH can be measured at 340 nm absorbance and GSNO reductase enzyme activity can be obtained by measuring the NADH value at a constant time interval at an absorbance of 340 nm (A340).

All reagents used were diluted in 100 mM sodium phosphate buffer containing 100 μM zinc chloride. For the measurement, 25 μl of 800 nM GSNO reductase and 25 μl of 4% DMSO solution were mixed in a 96-well plate and reacted at 30 ° C. for 10 minutes. Subsequently, the substrate solution (1 mM GSNO, 0.5 mM NADH) Was added. Thereafter, the absorbance at 340 nm was measured at intervals of 20 seconds for 5 minutes to obtain a Vmax value. The Vmax values were calculated using Kinetics of Flexstation SoftMax pro software (Molecular device). As a positive control for this experiment, 1 mu M of N6022 compound known to inhibit the activity of GSNO reductase was used. The structure of the N6022 compound is shown in the following formula (1).

The degree of inhibition of GSNO reductase activity by N6022 was measured by comparing the Vmax value of the negative control (DMSO) (Fig. 1c). GSNO reductase activity of 100 kinds of native plants in Jeju area was measured and shown in FIG.

Five extracts showed 85% or more inhibitory activity against GSNO reductase activity. The extracts of leaf and egg plant showed inhibition of GSNO activity by 97.0%.

In order to measure the IC50 values of the mixed extracts, leaves and branches of P. falciparum leaves and branches, the final concentrations of extracts and fractions were varied in the range of 3 μg / mL to 200 μg / mL, and the inhibitory effect of GSNO reductase And the results are shown in [Table 2] and [Figure 3]

The IC50 (unit: 쨉 g / mL) division  GSNO reductase (IC50) extract 17.387 Leaf Extract: n-Hexane Fraction 102.971 Leaf Extract: Ethyl Acetate Fraction 6.562 Leaf Extract: Butanol fraction 14.126 Leaf Extract: Water Fraction 27.136 Eggplant extract: n-hexane fraction 31.910 Eggplant extract: ethyl acetate fraction 5.954 Branch Extract: Butanol fraction 9.637 Eggplant extract: water fraction 36.716

As shown in the above Table 2, the extracts and fractions of Alnus japonica have GSNO reductase inhibitory activity, and the IC50 value of the ethyl acetate fraction of the eggplant extract was lowest at 5.954.

<Experimental Example 2> Measurement of PDE4A1 activity

To determine the activity of PDE4A1, cAMP levels were quantitated using time resolved fluorescence resonance energy transfer immunoassay. The TR-FRET method is based on the fact that if the distance between the energy donor europium (Eu) chelate-labeled cAMP tracer and the energy-accepting Ulight-labeled cAMP antibody is shortened, Energy is transferred. This method utilizes the competitive inhibition between Ulight-anti-cAMP and free-cAMP. When the level of free cAMP is greatly degraded by PDE4A1, the fluorescence resonance energy transfer increases, while PDE4A1 activity is inhibited Increased levels of free cAMP result in a decrease in fluorescence resonance energy transfer due to competitive inhibition between free cAMP and Ulight-labeled cAMP.

The reagent used was LANCE Ultra cAMP kit (PerkinElmer) and PDE4A1 enzyme was purchased from BPS Bioscience. 2.5 μL of 0.8 μg / μL PDE4A1 diluted in an enzyme reaction buffer (1 × HBSS, 5 mM HEPES, pH 7.4, 3 mM MgCl 2, 0.1% BSA) and 2.5 μL of a 4% DMSO solution were reacted for 5 minutes and then 5 μL of 12 nM cAMP Were mixed and further reacted for 1 hour. At this time, Rolipram (Sigma) having a final concentration of 20 μM can be used as a reference compound. After the enzymatic reaction, fluorescence resonance resonance was measured by adding Eu-cMAP tracer diluted 1/50 and 1/150 and Ulight-anti-cAMP to stop / detection buffer (1 mM IBMX, Sigma) 1 hour later, the activity of PDE4A1 was measured by measuring 340 nm excitation and 665 nm emission values through an Envision plate reader (PerkinElmer). A 384-well small volume microplate (Greiner) was used in consideration of the final volume of 20 μl.

 It was confirmed that each extract of 100 kinds of land plants of Jeju Island can inhibit the activity of PDE4A1. The activity of PDE4A1 reacted with DMSO was set at 100, and five kinds of extracts having 85% or more PDE4A1 activity inhibitory effect among 100 kinds of extracts were selected as effective extracts. The results are shown in FIG. Of these, 71 is a timber extract. The extracts of P. vivax showed 83.2% inhibition of PDE4A1 activity.

The inhibitory effect of PDE4A1 was measured by varying the final concentrations of extracts and fractions in the range of 3 μg / mL to 200 μg / mL in order to measure the IC50 values of the fractions of Alnus japonica extract, And Fig. 5, respectively.

The IC50 (unit: 쨉 g / mL) division PDE4A1 (IC50) extract 4.4 Leaf Extract: n-Hexane Fraction 180.9 Leaf Extract: Ethyl Acetate Fraction 2.4 Leaf Extract: Butanol fraction 4.9 Leaf Extract: Water Fraction 13.7 Eggplant extract: n-hexane fraction 10.0 Eggplant extract: ethyl acetate fraction 1.6 Branch Extract: Butanol fraction 5.5 Eggplant extract: water fraction 8.0

As shown in Table 3, the extracts and fractions of Alaskan pollack had PDE4A1 inhibitory activity, and the IC50 value of ethyl acetate fraction of Alaska pollack was the lowest at 1.6.

As can be seen from the above results, P. japonicus extract has excellent inhibitory activity against GSNO reductase and PDE4A1, which can be useful for prevention and treatment of asthma.

Claims (4)

A composition for the prevention and treatment of asthma comprising as an active ingredient an Allium cepa extract.
The method according to claim 1,
The composition for preventing and treating asthma according to any one of (I) to (V) below.

(I) Extracts obtained by extracting leaves, branches or mixtures thereof from water with water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof;
(II) a hexane fraction obtained by suspending water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, in a distiller's water, and sequentially fractionating the mixture with hexane, ethyl acetate and butanol;
(III) Ethyl acetate fractions obtained by suspending a mixture of water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof with distilled water, and sequential fractionation with hexane, ethyl acetate and butanol;
(IV) a butanol fraction obtained by suspending a mixture of water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof with distilled water, and sequentially fractionating the mixture with hexane, ethyl acetate and butanol; And
(V) Residual layer fraction obtained by suspending water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, in a mixture of water and a mixture of wood, leaves or a mixture thereof, in distilled water and sequentially fractionating the mixture with hexane, ethyl acetate and butanol.
3. The method according to claim 1 or 2,
Wherein the composition is a pharmaceutical composition.
3. The method according to claim 1 or 2,
Wherein the composition is a food composition.

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