WO2021167309A1 - Composition de suppression de l'asthme contenant une aldéhyde déshydrogénase - Google Patents

Composition de suppression de l'asthme contenant une aldéhyde déshydrogénase Download PDF

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WO2021167309A1
WO2021167309A1 PCT/KR2021/001934 KR2021001934W WO2021167309A1 WO 2021167309 A1 WO2021167309 A1 WO 2021167309A1 KR 2021001934 W KR2021001934 W KR 2021001934W WO 2021167309 A1 WO2021167309 A1 WO 2021167309A1
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asthma
saccharomyces cerevisiae
aldh
present
kwonp
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PCT/KR2021/001934
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English (en)
Korean (ko)
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권흥택
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주식회사 피코엔텍
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/14—Yeasts or derivatives thereof
    • A23L33/145—Extracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/062—Ascomycota
    • A61K36/064—Saccharomycetales, e.g. baker's yeast
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/18—Baker's yeast; Brewer's yeast

Definitions

  • the present invention relates to an asthma inhibitory composition comprising an aldehyde dehydrogenase. More specifically, the present invention provides Saccharomyces cerevisiae KwonP-3 and Saccharomyces cerevisiae KwonP-3) with accession number KCTC13925BP (Saccharomyces cerevisiae KwonP-1), KCTC14122BP ( Saccharomyces cerevisiae KwonP-2), KCTC14123BP (Saccharomyces cerevisiae KwonP-3) isolated from strains.
  • KCTC13925BP Saccharomyces cerevisiae KwonP-1
  • KCTC14122BP Saccharomyces cerevisiae KwonP-2
  • KCTC14123BP Sacharomyces cerevisiae KwonP-3 isolated from strains.
  • Asthma is a chronic disease that causes respiratory distress symptoms due to bronchial hypersensitivity and intermittent airway constriction due to chronic inflammation in the airways. It is known that there is no cure yet.
  • the antibodies recognize it and activate the immune system, leading to an inflammatory response that causes inflammation, swelling, and constriction of the airways. do.
  • Th2 lymphocytes in asthmatic patients are activated to increase IL-4, IL-5, IL-9 or IL-13.
  • IL-4 promotes mucus production and B-cell proliferation, and causes dyspnea. It produces IgE, which activates mast cells that secrete inflammatory mediators such as histamine or leukotriene, and expresses VCAM-1, which delivers eosinophils to lung tissue.
  • IgE which activates mast cells that secrete inflammatory mediators such as histamine or leukotriene
  • VCAM-1 which delivers eosinophils to lung tissue.
  • typical respiratory symptoms of asthma such as shortness of breath, cough, sputum, and wheezing (yin)
  • atypical symptoms may also appear.
  • Asthma sufferers may also experience elevated heart rate, dry crackling, etc.
  • cyanosis due to lack of oxygen, strong chest pain, numbness in limbs or sweating in the palms of the palms may be felt just before loss of consciousness, which is common Severe asthma attacks that do not respond to treatment can be life-threatening, leading to respiratory arrest and death.
  • This bronchial asthma is a very common disease that is reported to be suffering from about 5 to 10% of the total population in most countries around the world, including Korea. The prevalence is increasing worldwide.
  • External factors include airborne pollutants, various allergens and industrial dust. These asthma-inducing substances produce IgE in an immune response, and the produced IgE blocks the nasal mucosa, skin, and airways, and binds to receptors on the surface of mast cells in the bronchi to cause inflammation.
  • 'Treg cells' regulatory T cells
  • Asthma also has genetic factors. Human chromosome 5, ⁇ 2-adrenoceptor gene, and human chromosome 11, 12, 14, and 16 are involved, and it is reported that changes in these genes cause an increase in IgE and hypersensitivity of the bronchial tubes.
  • bronchial asthma When the nasal mucosa or bronchial mucosa is stimulated by bronchial asthma triggers, an excessive immune response (allergic reaction) occurs and inflammation occurs in the airway mucosal tissue, and accordingly, the three major symptoms of bronchial asthma: cough, aerobic wheezing, and dyspnea. This causes bronchial asthma and rhinitis symptoms such as runny nose, sneezing, or stuffy nose.
  • ROS reactive oxygen species
  • Treatment methods for bronchial asthma include avoidance and desensitization to remove or avoid allergens that cause bronchial asthma, drug therapy to select drugs, or immunotherapy to immunize by injecting a small amount of an allergen. .
  • bronchial asthma used in the drug therapy
  • a bronchodilator as a ⁇ 2-agonist that improves symptoms by widening a constricted airway
  • a corticosteroid-based inhalant for symptom relief or an anti-allergic agent
  • some asthma treatment drugs such as water-soluble IL-4 receptor, water-soluble IL-13 and anti-IL-5 antibody using a mechanism to inhibit the action of Th2-type cytokines are being developed and sold.
  • asthma medications can be purchased only through a doctor's consultation and prescription, and since most of them are steroid-based drugs, there is a problem that side effects may occur when used for a long period of time.
  • aldehyde dehydrogenase extracted from yeast inhibits cell infiltration and interleukin-4 (interleukin-4).
  • -4, IL-4) and interleukin-13 IL-13
  • Interleukin 4 (hereinafter, abbreviated as IL-4) secreted from T cells is a cytokine of about 20 kD consisting of 129 amino acids, and is an important cytokine that increases B-cell differentiation factors and B-cell proliferation factors, especially IgE. .
  • IL-4 is initially secreted by basophils and additionally secreted by activated T-cells (TH2). It has been reported to contribute to B cell activation, IgE type conversion, and induction of Th2 differentiation.
  • IL-4 production by Th2 cells is also accelerated by IL-4, and the receptor for IL-4 is a 130 kD protein that binds two cysteines, IL-4R ⁇ + ⁇ b and IL-4R ⁇ +IL-13R ⁇ .
  • IL-4 has many biological functions, such as stimulating activated B cells and T cells and differentiating B cells into plasma cells, and is an important regulator of humoral immunity and adaptive immunity.
  • IL-4 is known to induce class switching from B progenitor cells to B cells capable of producing IgE and IgG4, as well as promote the production of MHC class II. It causes an increase in IgE, resulting in an immediate onset hypersensitivity allergic reaction. IL-4 acts synergistically with IL-13 and is the causative agent of IgE- or eosinophil-mediated inflammatory responses.
  • IgE antibody When an IgE antibody is produced by exposure to an antigen, IgE binds to the surface of mast cells or basophils, and when the IgE-bound mast cells are exposed to the same antigen a second time, degranulation of the mast cells or basophil intragranule occurs and vasoactive substances Phosphorus histamine, serotonin, chemotactic factors of eosinophils or neutrophils, leukotrienes, and prostaglandins are released.
  • the ALDH-containing composition of the present invention can be an asthma treatment with small side effects by inhibiting IgE increase through IL-4 or IL-13 inhibition.
  • Interleukin 13 is a protein consisting of 122 amino acids, and the receptor for IL-13 is CD132 ( ⁇ c).
  • IL-13 is functionally similar to IL-4 and is secreted from Th2 cells, dendritic cells, and mast cells. alleviate
  • the receptor for IL-13 is composed of IL-13R ⁇ and IL-4R ⁇ , the IL-13 receptor also responds to IL-4.
  • the receptor for Il-13 is expressed in human B cells, but not in mouse B cells. IL-13 also differentiates monocytes into dendritic cells. However, it has been reported that IL-13 gene inactive mice do not actively produce Th2 cells or IgE.
  • IL-13 inhibitors or IL-13 signaling blockers can also be used as therapeutic agents for immune diseases such as atopy or asthma.
  • the aldehyde dehydrogenation reaction is a reaction for changing acetaldehyde or various aldehydes present in human metabolites into organic acids that can be discharged as waste products.
  • the enzyme that promotes this aldehyde dehydrogenation reaction decomposes the aldehyde, which is toxic to cells, and relieves carbonyl stress applied to cells and tissues.
  • aldehyde dehydrogenases A series of proteins containing functional groups of enzymes involved in aldehyde dehydrogenation are called aldehyde dehydrogenases, and about 20 types of ALDH are known so far. Therefore, in the present specification, the scope of the term ALDH includes all of the 20 known types of ALDH, all fusion proteins containing all or part of the dehydrogenation functional groups of enzymes involved in aldehyde dehydrogenation, and enzymes involved in aldehyde dehydrogenation. All recombinant proteins containing some or all of the dehydrogenation functional groups are included.
  • This prior art describes in detail mitochondrial aldehyde dehydrogenase-2 (ALDH2), a tetrameric protein consisting of four identical subunits encoded in the nuclear genome and transported into the mitochondria, each of 500 amino acid residues. wherein the tetramer is a dimer of a dimer, wherein the interface between the monomers forming the dimer is different from the interface between the two dimers forming the tetramer, and each subunit has three major sites, i.e., an aldehyde Dehydrogenation catalytic sites, coenzyme or NAD+-binding sites, and oligomerization sites are disclosed.
  • ADH2 mitochondrial aldehyde dehydrogenase-2
  • ALDH2 related diseases and conditions include ischemic stress, chronic free-radical related diseases, acute free-radical related diseases, insensitivity to nitroglycerin (eg, in angina and heart failure), hypertension, diabetes. , osteoporosis, cancer, Fanconi anemia, Alzheimer's disease, Parkinson's disease, alcoholism, alcohol intolerance, alcohol addiction, alcohol abuse disease, alcohol intoxication, alcohol dependence, alcohol poisoning
  • modulators of ALDH2, agonists of ALDH2, or antagonists of ALDH2, including symptoms of alcohol consumption, and drug addiction include ischemic stress, chronic free-radical related diseases, acute free-radical related diseases, insensitivity to nitroglycerin (eg, in angina and heart failure), hypertension, diabetes. , osteoporosis, cancer, Fanconi anemia, Alzheimer's disease, Parkinson's disease, alcoholism, alcohol intolerance, alcohol addiction, alcohol abuse disease, alcohol intoxication, alcohol dependence, alcohol poisoning
  • An agonist of ALDH2 is also useful for reducing the level of a compound that generates an aldehyde substrate of ALDH2 in a subject upon ingestion, absorption, or inhalation of a compound that generates an aldehyde substrate of ALDH2.
  • ALDH2 antagonists are used as adjuncts to standard cancer treatment. It has also been disclosed that antagonists of ALDH2 are useful in treating or suppressing alcoholism and drug addiction. However, this prior art does not make any suggestion or mention about the possibility that ALDH, which is the subject of the present invention, may inhibit the action of interleukin-4 or interleukin-13.
  • Korean Patent Application No. 10-2020-0019858 describes Saccharomyces cerevisiae that simultaneously produces aldehyde dehydrogenase (hereinafter abbreviated as ALDH) and glutathione, which are used as active ingredients in the pharmaceutical composition and food composition for inhibiting asthma of the present invention.
  • ALDH aldehyde dehydrogenase
  • glutathione glutathione
  • a basic object of the present invention is to provide a food composition containing an aldehyde dehydrogenase having an effect of alleviating and suppressing asthma symptoms.
  • Another object of the present invention is a composition for treating asthma and atopic rhinitis containing, as an active ingredient, a fusion protein or a recombinant protein comprising an aldehyde dehydrogenase having an effect of preventing and treating asthma symptoms or an action site involved in an aldehyde dehydrogenation reaction. is to provide
  • Saccharomyces cerevisiae yeast accession number KCTC13925BP (Saccharomyces cerevisiae KwonP-1), KCTC14122BP ( Saccharomyces cerevisiae KwonP-2), KCTC14123BP ( Saccharomyces cerevisiae KwonP-3) aldehyde isolated from It is achieved by providing a composition which contains a dehydrogenase and exhibits an asthma prevention or alleviation effect.
  • the yeast used by the present inventors for the production of ALDH for asthma suppression is Saccharomyces cerevisiae yeast, which is generally widely used for fermenting beer or bread. About 250 types of Saccharomyces cerevisiae yeast known to date have been reported, and all of them can be appropriately used for the ALDH production of the present invention.
  • the asthma inhibitory composition of the present invention is a novel Saccharomyces cerevisiae yeast deposited by the present inventors with the international depository KCTC, deposit number KCTC13925BP (Saccharomyces cerevisiae KwonP-1), KCTC14122BP ( Saccharomyces cerevisiae KwonP-1) cerevisiae KwonP-2) or KCTC14123BP ( Saccharomyces cerevisiae KwonP-3) may be used as a raw material for the production of the ALDH-containing composition for inhibiting asthma of the present invention.
  • KCTC13925BP Sacharomyces cerevisiae KwonP-1
  • KCTC14122BP Saccharomyces cerevisiae KwonP-1
  • KCTC14123BP Saccharomyces cerevisiae KwonP-3
  • the ALDH-containing compositions according to the present invention are effective in treating and preventing inflammatory reactions caused by oxidative stress, such as bronchial inflammation, allergic rhinitis, and asthma.
  • ALDH is not only contained in easily purchased foods, but also has the advantage of having little side effects on the human body because it is extracted from edible yeast.
  • the yeast extract of Saccharomyces cerevisiae according to the present invention can be used for immunoglobulin E (IgE) and total number of inflammatory cells and lymphocytes, monocytes, neutrophils, and eosinophils in the bronchoalveolar lavage fluid of asthma-induced mice in an in vivo experiment. It has the activity of inhibiting the increase and the secretion of airway mucus, and reduces the Th2 cytokines IL-4 and IL-13 and the degree of airway inflammation.
  • IgE immunoglobulin E
  • composition of the present invention exhibits an effect of preventing or treating bronchial asthma, allergic asthma, atopic asthma, non-topic asthma, exercise-induced asthma, aspirin asthma, psychogenic asthma, or alveolar asthma.
  • the ALDH contained in the composition of the present invention reduces airway hypersensitivity, suppresses the amount of active oxygen in the airways, or inhibits the infiltration of inflammatory cells in the bronchial tubes, thereby reducing the number of inflammatory cells in the bronchus, reducing the concentration of IgE antibody in the serum, or IL-4, a Th2 cytokine and IL-13, and the effect of inhibiting the expression of apoptosis-related proteins (JNK, ERK, p38, BCL-2 family, caspase-3, NF- ⁇ B) in the mechanism of regulating hypersensitive respiratory symptoms indicates Accordingly, the ALDH-containing compositions of the present invention can be used as pharmaceuticals and health foods useful for the prevention, improvement or treatment of bronchial asthma.
  • 1 is a graph showing the inhibitory effect of interleukin-4 increase in RBL-2HE cell line by the ALDH-containing composition of the present invention.
  • FIG. 2 is a graph showing the inhibitory effect of interleukin-13 in the sera of control and experimental mice of the ALDH-containing composition of the present invention.
  • FIG. 3 is a graph showing the IgE reduction effect of the ALDH-containing composition of the present invention in the sera of control and experimental mice.
  • ALDH-containing pharmaceutical composition or food composition of the present invention wild Saccharomyces yeast, Saccharomyces cerevisiae KwonP1 (KCTC13925BP), Saccharomyces cerevisiae KwonP2 (KCTC14122BP) or Saccharomyces cerevisiae Dry powder, lysate, or ALDH-containing extract powder of KwonP3 (KCTC14123BP) is included as an active ingredient.
  • Saccharomyces cerevisiae strain dry powder contained as an active ingredient in the composition of the present invention a method for preparing strains used for preparing ALDH-containing extract powder, lysate, Korea cited in the specification of the present invention It is described in detail in the specification of patent application No. 10-2020-00198858.
  • the ALDH-containing composition of the present invention may further include a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier includes, but is not limited to, saline, sterile water, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, and the like.
  • the ALDH-containing composition of the present invention may contain glutathione derived from Saccharomyces cerevisiae, and may be formulated as an aqueous solution, suspension, emulsion, etc. by additionally adding a diluent, dispersant, surfactant, binder, lubricant, etc. , pills, capsules, granules or tablets.
  • the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount.
  • the effective amount level can be determined according to the patient's disease type, severity, drug activity, sensitivity to drug, administration time, administration route and excretion rate, duration of treatment, factors including concurrent drugs, and factors well known to those skilled in the art.
  • the food composition of the present invention is characterized in that it is formulated with one selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquid formulations, including one or more of carriers, diluents, excipients, and additives.
  • Additives that may be further included in the food composition of the present invention include natural carbohydrates, flavoring agents, nutrients, vitamins, synthetic flavoring agents, natural flavoring agents, colorants, fillers, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH Regulators, stabilizers, preservatives, antioxidants, glycerin, alcohols and the like can be used.
  • Example 1-1 Yeast fermentation process in Saccharomyces cerevisiae containing ALDH
  • Saccharomyces cerevisiae yeast (KCTC139525BP) containing ALDH was fermented and cultured for 24 hours in an incubator at 160 rpm and 30 ° C using YPD medium (yeast extract, peptone, and glucose containing medium) in a 200 mL flask, This culture was carried out for 72 hours through a 5 L fermenter (Marado-05D-PS, CNS, Korea). After completion of the culture, the yeast was centrifuged using a high-speed centrifuge (Supra R22, Hanil, Korea).
  • YPD medium yeast extract, peptone, and glucose containing medium
  • Example 1-2 Preparation of ALDH-containing composition of the present invention
  • the centrifuged ALDH-containing yeast was frozen in a cryogenic freezer (CLN-52U, Nihon freezer, Japan) for 2 days, and then freeze-dried for 2 days in a freeze dryer (FDU-7006, Operon, Korea).
  • a freeze dryer FDU-7006, Operon, Korea.
  • PBS phosphate-buffered saline
  • 0.5 mm glass beads for cell disruption 11079105, Biospec 10 g was put into the bead homogenizer (Mixer Mill MM400, Retsch, Germany) to disrupt yeast over a total of 3 times for 2 minutes each.
  • After centrifugation using a high-speed centrifuge (Supra R22, Hanil, Korea), only the supernatant was separated and freeze-dried for 2 days with a freeze dryer (FDU-7006, Operon, Korea).
  • OVA Ovlabumin; mouse inflammation inducer
  • aluminum hydroxide immune response enhancer
  • the powder of the ALDH-containing composition of the present invention was dissolved in physiological saline from the time point after a week had elapsed and oral administration was carried out. caused It was conducted once a day for a total of 5 days.
  • the powder of the composition containing ALDH of the present invention was administered at 6 mg/kg/day, 30 mg/kg/day, and 150 mg/kg/day.
  • the powder of the ALDH-containing composition of the present invention was administered in the morning, and 20 ug/head/day of ovalbumin was administered by inhalation using a nebulizer in the afternoon. And this operation was repeated for 5 days. At this time, the powder administration of the ALDH-containing composition of the present invention was administered at different doses in 3 groups of 6 mg/kg, 30 mg/kg, and 150 mg/kg.
  • Dexamethasone (steroid) administration group was 1mg/kg/day per mouse.
  • control is a normal group to which ovalbumin, an asthma inducer, dexamethasone, a control drug for asthma treatment, or the powder of the composition containing ALDH of the present invention was not administered as a control group.
  • the OVA group was a group in which ovalbumin was administered intraperitoneally on the 1st and 8th days of the experiment and by inhalation using a nebulizer on the 14th day to induce asthma by the above method.
  • ovalbumin was administered intraperitoneally on the 1st and 8th days as described above, and from the 14th day onwards, dexamethasone (steroid) was administered 1mg/kg/day per mouse in the morning and repeated for 5 days from the 14th day, and ovalbumin 20ug/day in the afternoon.
  • the group was administered head/day by inhalation using a nebulizer.
  • the ALDH 6mg/kg group is a group in which 6mg/kg of the ALDH-containing composition powder of the present invention is administered in the morning repeatedly for 5 days from the 14th day, and 20ug/head/day of ovalbumin is administered by inhalation using a nebulizer in the afternoon, and ALDH
  • the 30mg/kg group is a group in which 30mg/kg of the ALDH-containing composition powder of the present invention is administered in the morning repeatedly for 5 days from the 14th day, and ovalbumin 20ug/head/day is administered by inhalation using a nebulizer in the afternoon
  • the /kg group is a group in which 150 mg/kg of the ALDH-containing composition powder of the present invention is administered in the morning repeatedly for 5 days from the 14th day, and 20 ug/head/day of ovalbumin is administered by inhalation using a nebulizer in the afternoon.
  • mice were sacrificed and Bronchioalveolar fluid (BALF) components were analyzed in the lung bronchi to measure the number of WBCs (white blood cells), and cytokines such as IL-4 and IL-13 were analyzed by enzyme-linked immunoassay (ELISA) method. was used for measurement.
  • BALF Bronchioalveolar fluid
  • nebulizer system After oral administration with the nebulizer system for 5 days, an autopsy was performed the next day. There were a total of 8 animals in one group, and blood was collected from all 8 mice. Bronchial Alveolar Lavage Fluid (BALF) was collected from 4 animals in each group, and the lungs were removed from the remaining 4 animals. The extracted lungs were divided into thirds. One was stored in 10% formalin to examine histopathology, and the other two were stored at -80°C to view cytokines.
  • BALF Bronchial Alveolar Lavage Fluid
  • the BALF obtained from the animal was centrifuged at 4°C at 3000 rpm for 5 minutes. Remove the supernatant and mix with PBS buffer to the remaining pellet. Thereafter, blood cell analysis was performed using a hemacytometer (Hemavet 950FS, Drew Scientific Inc, Korea).
  • the remaining BALF used for blood cell analysis was centrifuged at 4°C and 3,000 rpm for 5 minutes. After that, the supernatant was discarded, PBS buffer was added, and centrifugation was performed at 800 rpm for 5 minutes using a Cyto-Tek machine. Then, it was stained with Diff Qucik reagent.
  • BALF Bronchial Alveolar Lavage Fluid
  • Diff Qucik reagent is used using a hemocytometer (Hemavet 950FS, Drew Scientific Inc, Korea). Inflammatory cell count, neutrophil and mononuclear leukocytes were analyzed by staining using
  • An IgE ELISA set was used, and Capture Ab diluted in Coating Buffer was added to a micro well and incubated at 4 °C overnight. After washing 3 times, add assay diluent to each well and incubate at room temperature. After washing 3 times, add standard and sample diluted with assay diluent to each well and incubate at room temperature. After washing 5 times, put Working Detector in each well and incubate at room temperature. After washing 7 times, add Substrate Solution to each well and incubate in dark conditions at room temperature. Stop solution in each well was measured at 450 nm within 30 minutes.
  • Paraffin removal and hydration were carried out, and after dehydration, they were washed with tap water for 5 minutes, dried well, and then stained with heamatoxylin solution for 2 minutes. After staining, clarification and dehydration were performed, and tissue encapsulation was performed.
  • tissue encapsulation was performed.
  • Enzyme-Linked Immunosorbent Assay Add Capture Ab diluted in Coating Buffer to micro-wells and incubate at 4°C overnight. After washing 3 times, add assay diluent to each well and incubate at room temperature. After washing 3 times, add standard sample diluted with Assay Diluent to each well and incubate at room temperature. After washing 5 times, put Working Detector in each well and incubate at room temperature. After washing 7 times, add Substrate Solution to each well and incubate in dark conditions at room temperature. Stop solution in each well was measured at 450 nm within 30 minutes. Cytokine ELISA analysis was Th1 (IL-6, IFN- ⁇ ), Th2 (IL-4, IL-5, IL-13), Th17 (IL-6, TNF- ⁇ ) were analyzed.
  • Th1 IL-6, IFN- ⁇
  • Th2 IL-4, IL-5, IL-13
  • Th17 IL-6, TNF- ⁇
  • RT-PCR Real-time polymerase chain reaction
  • OVA ovalbumin
  • Al hydroxide immunophilicity enhancer
  • Example 5-1 Administration of the ALDH-containing composition of the present invention
  • Lyophilized ALDH-containing yeast lysate was dissolved in phosphate-buffered saline (PBS) to a concentration of 6 mg/kg, 30 mg/kg, and 150 mg/kg based on 560 ⁇ L, respectively, and then vortexer (F202A0175, VELP scientific, Italy) for 1 minute (C. Kla an. et al., J. Immunol, 1;199(1):48-61, 2017).
  • the positive control material, dexamethasone (Sinilpharm, Korea) was dissolved in physiological saline (Cleancle, JW Pharmaceutical, Korea) to a concentration of 1 mg/kg and mixed with a vortexer (F202A0175, VELP scientific, Italy) for 1 minute (C. Kla combined. et al., J. Immunol, 1;199(1):48-61, 2017).
  • Example 5-2 Administration of the ALDH-containing composition of the present invention to an asthma-induced mouse model
  • Example 6-1 Mouse autopsy and collection of blood and lung tissue
  • OVA spraying and oral administration using an ultrasonic nebulizer were performed for 5 days, and an autopsy was performed the next day.
  • Blood was collected from all 8 animals per group using a 1 mL syringe (Kovax-syringe 1 mL 26G 1/2, Koreavaccine, Korea), and lungs were removed from 4 mice.
  • the excised lungs were divided into thirds, one stored in 10% formalin to examine histopathology, and the other two pieces were stored at -80°C for interleukin measurement.
  • Example 6-2 Collection of bronchoalveolar lavage fluid
  • Example 7-2 In vitro efficacy of the ALDH-containing composition of the present invention for asthma
  • the RBL-2HE cell line was pre-treated with the ALDH-containing composition of the present invention at each concentration for 20 minutes, and an intracellular inflammatory state was induced by adding IgE and BSA to the cell line.
  • the inhibitory effect of the ALDH-containing composition of the present invention was measured by measuring the expression level of IL-4, a major marker of the asthma induction model, and the results are shown in FIG. 1 . Compared to the IL-4 expression level of the asthma-induced cell line, 13%, 34%, and 35% decrease in the IL-4 expression level was confirmed in the cells treated with the ALDH-containing composition of the present invention for each concentration.
  • the inhibitory effect of the ALDH-containing composition of the present invention on the IL-4 expression level was confirmed, and it was also confirmed that the IL-4 inhibitory effect increased depending on the dosage.
  • interleukin-13 showed up to about 80% reduction in expression at concentrations of 150 mg/Kg and 300 mg/Kg, which are the doses of the ALDH-containing composition of the present invention. (FIG. 2)
  • Example 7-3 IgE reduction effect of the ALDH-containing composition of the present invention in asthma-induced mice
  • the blood in a 1.5 mL tube was centrifuged at 12,000 rpm and 4° C. for 20 minutes, and only the supernatant was separated and used in the experiment for IgE measurement.
  • an IgE ELISA set (555248, BD bioscience, USA) was used, and the analysis method is the same as in Example 5-1.
  • the analysis results are shown in FIG. 3, and the IgE reduction effect was confirmed in the group administered with the ALDH-containing composition of the present invention compared to the asthma-induced mice.
  • mice Female and male ICR mice (7 weeks old) were received and acclimatized for 7 days. During the acclimatization period, general symptoms were observed, and only healthy animals were used for the test. Feed and water were ingested ad libitum, and group separation was performed so that there were 5 males and 5 females in each group based on the average body weight of about 20 g the day before oral administration.
  • the test substance was prepared by dissolving in physiological saline so that the doses of the test animals were 0, 750, 3000, and 5000 mg/Kg, respectively, based on the ALDH-containing composition of the present invention.
  • the standard of administration dose complied with the Korea National Toxicology Program (KNTP) toxicity test manual of the Ministry of Food and Drug Safety, and the maximum applied dose 5000mg/Kg guided by the KNTP manual was applied as the maximum concentration of this experiment.
  • Samples prepared for each group were orally administered once to each test animal, and physiological saline was administered to the normal group (G1).
  • Symptoms were observed at least once a day from the date of acquisition to the day of autopsy for all animals in the test group, and symptoms were observed for 7 days after oral administration. After the type of symptom observation, an autopsy was performed, and changes in each organ were visually observed at the time of autopsy.

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Abstract

La présente invention concerne une composition alimentaire ou pharmaceutique contenant un extrait de levure qui présente un effet de suppression des symptômes de l'asthme bronchique, l'asthme allergique, l'asthme atopique, l'asthme induit par l'exercice, l'asthme cardiaque ou l'asthme alvéolaire. Plus spécifiquement, la présente invention concerne un aliment ou une composition pharmaceutique supprimant l'asthme contenant une aldéhyde déshydrogénase qui est isolé à partir de souches de saccharomyces cerevisio ou numéro d'accès KCTC13925BP (Saccharomyces cerevisiae KwonP-1), KCTC14122BP (Saccharomyces cerevisiae KwonP-2), KCTC14123BP (Saccharomyces cerevisiae KwonP-3). [Dessin représentatif] figure 1
PCT/KR2021/001934 2020-02-18 2021-02-16 Composition de suppression de l'asthme contenant une aldéhyde déshydrogénase WO2021167309A1 (fr)

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KR1020210020186A KR102460545B1 (ko) 2020-02-18 2021-02-15 신규한 돌연변이 효모를 함유하는 천식 억제용 조성물

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011158689A1 (fr) * 2010-06-19 2011-12-22 天野エンザイム株式会社 Agent permettant de réduire l'acétaldéhyde dans la cavité orale
JP2013247939A (ja) * 2012-06-04 2013-12-12 Kiso Machi 新規酵母及びこれを含む医薬又は飲食品
KR20180003344A (ko) * 2016-06-30 2018-01-09 (주)아모레퍼시픽 효모 유래 세포밖 소포체를 포함하는 항염 조성물

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011158689A1 (fr) * 2010-06-19 2011-12-22 天野エンザイム株式会社 Agent permettant de réduire l'acétaldéhyde dans la cavité orale
JP2013247939A (ja) * 2012-06-04 2013-12-12 Kiso Machi 新規酵母及びこれを含む医薬又は飲食品
KR20180003344A (ko) * 2016-06-30 2018-01-09 (주)아모레퍼시픽 효모 유래 세포밖 소포체를 포함하는 항염 조성물

Non-Patent Citations (2)

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Title
BAZEWICZ CHRISTOPHER G.: "Aldehyde dehydrogenase in regulatory T‐cell development, immunity and cancer", IMMUNOLOGY, vol. 156, 1 January 2018 (2018-01-01), pages 47 - 55, XP055840160 *
FONSECA VANESSA M.B., MILANI THAMIRES M.S., PRADO RAFAEL, BONATO VANIA L.D., RAMOS SIMONE G., MARTINS FLAVIANO S., VIANNA ELCIO O.: "Oral administration of Saccharomyces cerevisiae UFMG A-905 prevents allergic asthma in mice : UFMG A-905 and asthma", RESPIROLOGY, vol. 22, no. 5, 1 July 2017 (2017-07-01), pages 905 - 912, XP055817368, ISSN: 1323-7799, DOI: 10.1111/resp.12990 *

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