WO2012144501A1 - 抗アレルギー物質、抗アレルギー剤及び食品 - Google Patents

抗アレルギー物質、抗アレルギー剤及び食品 Download PDF

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Publication number
WO2012144501A1
WO2012144501A1 PCT/JP2012/060389 JP2012060389W WO2012144501A1 WO 2012144501 A1 WO2012144501 A1 WO 2012144501A1 JP 2012060389 W JP2012060389 W JP 2012060389W WO 2012144501 A1 WO2012144501 A1 WO 2012144501A1
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WIPO (PCT)
Prior art keywords
strawberry
glyceraldehyde
food
allergy
phosphate dehydrogenase
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Ceased
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PCT/JP2012/060389
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English (en)
French (fr)
Japanese (ja)
Inventor
浩治 川原
井上　祐一
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Institute of National Colleges of Technologies Japan
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Institute of National Colleges of Technologies Japan
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Priority to EP12773893.8A priority Critical patent/EP2705851B1/en
Priority to US14/113,198 priority patent/US9511124B2/en
Publication of WO2012144501A1 publication Critical patent/WO2012144501A1/ja
Anticipated expiration legal-status Critical
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    • 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
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • 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
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
    • C12Y102/01012—Glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) (1.2.1.12)

Definitions

  • the present invention relates to an antiallergic substance, an antiallergic agent, and a food, and particularly relates to an antiallergic substance that functions as a physiologically active substance that suppresses IgE antibody production.
  • hay fever is said to be 15 to 20% of the whole population. Furthermore, it is said that this number will increase in the future, and it is getting worse.
  • the symptoms of hay fever are diseases that have a great impact on life such as runny nose, cough, and itchy eyes, but they are not fatal, so many depend on their patience, Although various treatments have been tried in hospitals, there is no decisive treatment, and palliative treatment with steroidal anti-inflammatory drugs is being performed when symptoms are severe.
  • This strong anti-inflammatory steroid-based anti-inflammatory agent has not only an anti-inflammatory action in the body but also a physiological action as a hormone because steroids are part of the hormone in the body, so there are also strong side effects. It has been pointed out. For this reason, it is important to use it under the strict supervision of a doctor, and many patients are concerned about the effects of such side effects.
  • functional foods do not aim to cure diseases like pharmaceuticals, but by incorporating them into daily diet, they can easily prevent disease and alleviate disease symptoms, thus improving the quality of life of patients. There are great expectations as a means to make this possible.
  • the inventors of the present application have constructed an experimental system that can reproduce the production of IgE antibody outside the body by taking out the immune cells involved in IgE antibody production outside the body and culturing the cells.
  • the inventors of the present invention have independently constructed a culture system using human peripheral blood lymphocytes that models the pharmacokinetics of human allergies, and have identified components exhibiting antiallergic effects from food components. Unlike the conventional method of testing using a large amount of experimental animals such as mice, this technique is extremely efficient because hundreds of specimens can be tested simultaneously in about 10 days of culture.
  • a human cell is used, if an effective component in the searched factor is found, it is highly possible that the effect is exhibited in the living body, and the time required for searching can be shortened.
  • it is possible to elucidate the action mechanism of the factor at the cellular level and thus has many excellent advantages such as enabling scientific evaluation of food functions (non- Patent Document 1).
  • the inventors of the present application have constructed the above-described experimental system, and were able to screen for substances having an effect of suppressing IgE production using this experimental system.
  • the present invention identifies a highly safe substance that can treat allergic diseases and allergic symptoms by suppressing IgE production, and does not cause side effects such as steroids and can be taken for a long time.
  • An object of the present invention is to provide an antiallergic substance containing the substance, and further to provide an antiallergic agent (pharmaceutical) containing the identified substance as a composition and a food called a functional food having an antiallergic effect.
  • the inventors of the present invention screen for substances having an effect of suppressing IgE production by adding various strawberry extracts into the culture system using an IgE antibody production experiment system.
  • I did the research.
  • the crude extract liquid of strawberry has an IgE antibody production inhibitory effect.
  • glyceraldehyde 3-phosphate dehydrogenase whose component showing the strongest activity is one of the metabolic enzymes by the purified fraction. (GAPDH).
  • the first aspect of the present invention is characterized in that glyceraldehyde 3-phosphate dehydrogenase is included as a component for suppressing the production of IgE antibodies as an antiallergic substance.
  • the component is provided as a food because it is expected to enable simple intake in daily life.
  • Strawberry which was the basis for the discovery of this ingredient, is one of the most popular foods in the market, but it is also used in raw and processed foods (drinks, strawberries, ice confectionery, etc.). This is particularly advantageous for food applications.
  • a second aspect of the present invention is characterized in that the composition containing glyceraldehyde 3-phosphate dehydrogenase is in the form of food.
  • the third aspect of the present invention enables glyceraldehyde triphosphate dehydrogenase to be used for allergy prevention and treatment by containing it as an active ingredient of an antiallergic agent.
  • glyceraldehyde 3-phosphate dehydrogenase having an antiallergic effect is contained not only in strawberries but in all living things.
  • this enzyme is known as an enzyme involved in the metabolism of organisms, it was not captured in terms of antiallergic effects as found in this application, but it is contained in a wide range of other organisms. Is also considered to have an antiallergic effect as a component for suppressing the production of IgE antibodies. Therefore, even in the case of food, the glyceraldehyde 3-phosphate dehydrogenase is not necessarily limited to those derived from strawberries, but may be derived from any suitable food component.
  • the antiallergic agent is an orally administered drug, it may tend to be selected from the same viewpoint as food, but it can be used for each product, including cosmetics that are included as antiallergic substances. In addition, it is only necessary to select those derived from appropriate organisms from a wide range of organisms.
  • glyceraldehyde 3-phosphate dehydrogenase was identified as one of the components having an antiallergic effect of suppressing IgE production, so that allergic diseases can be treated and allergic symptoms can be alleviated, and An antiallergic substance having high safety that does not cause side effects like steroids and can be taken for a long period of time is obtained, and an antiallergic agent (medicine) and a functional food can also be obtained.
  • the glyceraldehyde 3-phosphate dehydrogenase according to the present invention is an enzyme protein possessed by various organisms, and its amino acid composition is not necessarily required to be completely identical with the amino acid composition of strawberry-derived glyceraldehyde 3-phosphate dehydrogenase. do not do.
  • Strawberries are generally eaten raw and are also used in processed foods. Therefore, even if ingested, there is no adverse effect on the human body, and it is recognized that the food is highly safe. From this fact, the strawberry extract mainly composed of glyceraldehyde 3-phosphate dehydrogenase used in the present invention can be safely ingested.
  • the glyceraldehyde 3-phosphate dehydrogenase used in the present invention can be obtained by extracting strawberry pulp such as “Amaou” and “Toyonoka” with an aqueous solvent. Considering that the extract can be used for foods and cosmetics, it is preferable to use an aqueous solvent in terms of safety.
  • the intake concentration of the ingredients should be determined individually according to the age, sex, medical condition, etc. of the allergic patient, but is preferably 5% to 100% by weight with respect to the composition.
  • the composition of the present invention can be used, for example, in medicines and foods.
  • oral administration is preferred. Therefore, it can be formulated by mixing with sorbitol, gelatin, lactose, glucose, starch, citric acid, etc., which are generally used as pharmaceutical preparation additives.
  • the composition of the present invention can be blended into foods having a solid, liquid, gel or other shape. Since the blending can be performed by any known production method, for example, it can be blended in ice cream, chocolate, candy, soft drinks, and the like.
  • peripheral lymphocytes In order to obtain human peripheral lymphocytes, healthy human peripheral blood was separated by density gradient centrifugation. First, peripheral blood is collected from a healthy person in a vacuum tube containing heparin, and 5 ml of peripheral blood is collected on a 4 ml blood separation agent (Ficoll; GE Healthcare) previously dispensed into a 15 ml centrifuge tube. The blood was overlaid and centrifuged at 400 ⁇ g for 30 min. The uppermost plasma layer was obtained and stored at ⁇ 24 ° C.
  • a 4 ml blood separation agent Ficoll; GE Healthcare
  • lymphocytes between the plasma layer and the Ficoll layer were collected, washed with a basic synthetic medium ERDF (Kyokuto Seiyaku), and centrifuged and washed three times under conditions of 400 ⁇ g and 5 min.
  • the obtained lymphocytes were stored frozen at ⁇ 85 ° C., and after thawing, the lymphocytes were washed with ERDF medium and used.
  • the main functions of the added immunostimulant are adjuvant activity that enhances the immune response to the antigen by MDP, promotion of T cell proliferation and differentiation by IL-2, induction of IgE class switch by IL-4, and IL-6 This is induction of B cell differentiation.
  • 200 ml of human peripheral blood lymphocytes added with these immunostimulants and Cryj1 were dispensed into 96-well plates, and cultured in a 5% CO 2 incubator at 37 ° C. for 10 days to induce IgE antibody production.
  • the human plasma and human peripheral blood lymphocytes contained in this culture system were the same individuals.
  • the culture supernatant containing IgE antibody to be quantified was then allowed to stand at 37 ° C. for 1 hour, diluted 1/8 with 1% BSA / PBS, and dispensed at 50 ml / well.
  • 50 ml / well of a standard solution serially diluted from 1 mg / ml to 1/3 by 1/38 mg / ml was dispensed and allowed to stand at 37 ° C. for 1 hour.
  • biotin-labeled goat anti-human IgE antibody Biosource
  • Biosource biotin-labeled goat anti-human IgE antibody
  • streptavidin labeled with horseradish peroxidase (Funakoshi) was diluted 1,000 times with 1% BSA / PBS solution, dispensed at 100 ml / well, and allowed to stand at 37 ° C. for 1 hour.
  • 0.006% H2O2-0.2M citrate buffer (pH 4.0), 6 mg / ml ABTS- (NH4) 2 (Wako) (Wako)
  • ultrapure water was mixed at a ratio of 10: 1: 9, respectively, as a color developing solution.
  • FIG. 1 is a graph showing the results of confirming the effect of suppressing the IgE antibody production of strawberry varieties “Amaou” and “Toyonoka”.
  • FIG. 1 shows the result of confirming the antiallergic effect in this way.
  • glyceraldehyde 3-phosphate dehydrogenase is a metabolic enzyme widely possessed by living organisms, it is derived from a strawberry refined product using a rabbit-derived glyceraldehyde 3-phosphate dehydrogenase preparation that is commercially available as a general reagent.
  • the activity was compared with that of glyceraldehyde 3-phosphate dehydrogenase, it was confirmed that it had the same IgE production inhibitory effect.
  • the glyceraldehyde 3-phosphate derived from rabbit has an upper limit value and a lower limit value of 0 for comparison because the glyceraldehyde 3-phosphate concentration in the purified strawberry is in the range of 0.1 to 1 microgram / ml. The case where 1 microgram / ml and 1 microgram / ml were added was verified.
  • FIG. 2 is a graph showing a comparison of the IgE antibody production inhibitory action between purified strawberry and rabbit-derived glyceraldehyde 3-phosphate. The results confirmed in this way are shown in FIG.
  • the control in FIG. 2 is a comparative control, and the purified product is the purified strawberry product of the present invention.
  • the vertical axis represents the production amount of IgE antibody.
  • the strawberry purified product showed 140 nanograms / milliliter of antibody production suppression effect of about 20% or more.
  • Rabbit-derived glyceraldehyde triphosphate also showed an antibody production-suppressing effect of about 20% with approximately 140 nanograms / milliliter within the range of the added concentration. was recognized.
  • Atopic dermatitis model mice were induced with dermatitis by application of Picryl Chloride. That is, 5 g of Picryl Chloride was put in a conical flask, 40 ml of 100% ethanol was added, and the flask was slowly warmed and gently mixed to melt Picryl Chloride. Next, when the solution turned yellow, the heating was stopped, 10 ml of ice-cold distilled water was added, and the flask was quickly placed in ice, followed by crystallization of Picryl Chloride. The supernatant was passed through a glass filter (pore size: 20-30 ⁇ m), and the remaining crystals were collected.
  • the collected crystals were suspended in 200 ml of 50% ethanol, allowed to stand for several minutes, and then passed through a glass filter washed with 100% ethanol to collect the remaining crystals.
  • the collected crystals were suspended in 200 ml of 50% ethanol, allowed to stand for several minutes, and then passed through a glass filter washed with 100% ethanol to collect the remaining crystals.
  • the obtained crystal was placed on the filter paper, and the filter paper was folded so that the crystal was inside.
  • the filter paper was covered with aluminum foil to shield it from light, and dried at room temperature until the next morning. This preparation is dissolved in olive oil as a refined product of Picryl Chloride so as to be 0.8 to 1.0%, and a part of the body of the mouse is shaved and applied to the exposed skin. Onset induction was performed.
  • FIG. 3 is a graph showing the antiallergic effect of the strawberry composition using atopic dermatitis model mice. The result is shown in FIG. In the graph of FIG. 3, the horizontal axis indicates the number of days of intake of the strawberry extract, and the vertical axis indicates the dermatitis inflammation score. The higher the score, the higher the degree of inflammation. Although both the control experiment and the strawberry ingestion group showed similar trends in the degree of inflammation as a whole, the strawberry ingestion group has always had a lower inflammation score than the control experiment since about 21 days after ingestion. Therefore, symptom relief of inflammation by strawberry intake was observed.
  • FIG. 4 is a verification photograph of the antiallergic effect of the strawberry composition using atopic dermatitis model mice and rhinitis model mice.
  • 4A is a photograph of an atopic dermatitis model mouse
  • FIG. 4B is a photograph of a rhinitis model mouse.
  • Each symptom of the photograph on the left improved as shown in the photograph on the right by eating the strawberry extract.
  • Atopic dermatitis model mice show that dermatitis is reduced by eating strawberries.
  • allergic rhinitis model mice hair loss was observed around the nose of the control group, whereas in the strawberry extract intake group, rhinitis did not develop and consequently no hair loss around the nose. I understood.
  • the food having an antiallergic effect may be any food containing glyceraldehyde 3-phosphate dehydrogenase derived from various organisms (plants / animals) and suppressing IgE antibody production, such as strawberries. It can be applied not only to the improvement of varieties, but also to the improvement of varieties of other existing fruits and vegetables, and to new varieties that have been improved to have various characteristics expected in the future. This greatly contributes to foods having an antiallergic effect in the functional food field.

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  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
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  • Wood Science & Technology (AREA)
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  • General Engineering & Computer Science (AREA)
  • Polymers & Plastics (AREA)
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PCT/JP2012/060389 2011-04-22 2012-04-17 抗アレルギー物質、抗アレルギー剤及び食品 Ceased WO2012144501A1 (ja)

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EP12773893.8A EP2705851B1 (en) 2011-04-22 2012-04-17 Anti-allergy substance, anti-allergy agent, and food
US14/113,198 US9511124B2 (en) 2011-04-22 2012-04-17 Anti-allergic substance, anti-allergic agent, and food

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JP2011096512A JP5804592B2 (ja) 2011-04-22 2011-04-22 抗アレルギーのための医薬品組成物
JP2011-096512 2011-04-22

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2012233873A (ja) * 2011-04-22 2012-11-29 Institute Of National Colleges Of Technology Japan 抗アレルギー因子のスクリーニング方法
JP2015107931A (ja) * 2013-12-04 2015-06-11 株式会社バイオサイエンスリンク グリセルアルデヒド３リン酸脱水素酵素（ｇａｐｄｈ）由来ペプチド及びこれを含む抗アレルギー組成物
TWI619507B (zh) * 2013-03-22 2018-04-01 東宇生物科技股份有限公司 治療或預防過敏性疾病的有效成分

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WO2024106446A1 (ja) * 2022-11-15 2024-05-23 株式会社ニッスイ アレルギー性鼻炎またはアレルギー性結膜炎の症状の軽減または予防用組成物

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JP2000139404A (ja) * 1998-03-29 2000-05-23 Nippon Meat Packers Inc 低アレルゲン化食肉製品及びその製造方法

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AKIRA IWAMOTO ET AL.: "3J14p10 Ichigo Chushutsubutsu no IgE Kotai Sansei Yokusei no Mechanism", JAPAN SOCIETY FOR BIOSCIENCE, BIOTECHNOLOGY, AND AGROCHEMISTRY 2011 NENDO TAIKAI KOEN YOSHISHU, vol. 2011, 5 March 2011 (2011-03-05), pages 215, XP008171359 *
AKIRA IWAMOTO ET AL.: "AOlp In vitro Allergy Model ni Okeru Ichigo Chushutsubutsu no Ko- Allergy Sayo", JAPAN SOCIETY FOR BIOSCIENCE, BIOTECHNOLOGY, AND AGROCHEMISTRY, CHUSHIKOKU NISHI NIPPON SHIBU, JAPAN SOCIETY OF NUTRITION AND FOOD SCIENCE, KYUSHU - OKINAWA SHIBU, THE JAPANESE SOCIETY FOR FOOD SCIENCE AND TECHNOLOGY, NISHI NIPPON SHIBU, 2009 NENDO G, 30 October 2009 (2009-10-30), pages 59, XP008171371 *
AKIRA IWAMOTO ET AL.: "D02a Ichigo Chushutsubutsu Chu no Ko-Allergy Inshi", HEISEI 23 NENDO JAPAN SOCIETY FOR BIOSCIENCE, BIOTECHNOLOGY, AND AGROCHEMISTRY, NISHI NIPPON SHIBU · CHUSHIKOKU SHIBU GODO TAIKAI KOEN YOSHISHU, 16 September 2011 (2011-09-16), pages 54, XP008171370 *
ELSEVIER, J. IMMUNOL. METHODS, vol. 233, 2000, pages 33 - 40
HIKARU YOSHIDA: "Mechanism analysis of food components with anti-allergic function", HEISEI 23 NENDO SENKOKA TOKUBETSU KENKYU KOKAI HAPPYOKAI YOSHISHU, January 2012 (2012-01-01), XP055135840, Retrieved from the Internet <URL:http://www.kct.ac.jp/data/files/senkouka/tokubetu/080_Material_0l.pdf,http://www.kct.ac.jp/data/files/senkouka/tokubetu/03_Contents_0l.pdf,http://www.kct.ac.jp/senkouka/tokubetsu.html> *
ITOH, T. ET AL.: "Inhibitory effects of flavonoids isolated from Fragaria ananassas Duch on IgE-mediated degranulation in rat basophilic leukemia RBL-2H3", BIOORGANIC & MEDICINAL CHEMISTRY, vol. 17, no. 15, 1 August 2009 (2009-08-01), pages 5374 - 5379, XP026336545 *
IWAMOTO, A. ET AL.: "Purification and identification of an IgE suppressor from strawberry in an in vitro immunization system", CYTOTECHNOLOGY, vol. 64, no. 3, February 2012 (2012-02-01), pages 309 - 314, XP055134223, Retrieved from the Internet <URL:http://www.springerlink.com/content/51r3228636317105> *
KHAN, A.A. ET AL.: "Molecular cloning, characterization, and expression analysis of two class II chitinase genes from the strawberry plant", PLANT SCIENCE, vol. 166, no. 3, March 2004 (2004-03-01), pages 753 - 762, XP055134220 *
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See also references of EP2705851A4 *
VAN DER VENTEL, M.L. ET AL.: "Differential responses to natural and recombinant allergens in a murine model of fish allergy", MOLECULAR IMMUNOLOGY, vol. 48, no. 1, January 2011 (2011-01-01), pages 637 - 646, XP027578535 *
YOSHIHISA TAKAHATA ET AL.: "Identification of Chicken Meat Proteins Detected with IgE Antibodies from Allergic Patients", JAPANESE POULTRY SCIENCE, vol. 37, no. 4, 2000, pages 228 - 233, XP008171248 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2012233873A (ja) * 2011-04-22 2012-11-29 Institute Of National Colleges Of Technology Japan 抗アレルギー因子のスクリーニング方法
TWI619507B (zh) * 2013-03-22 2018-04-01 東宇生物科技股份有限公司 治療或預防過敏性疾病的有效成分
JP2015107931A (ja) * 2013-12-04 2015-06-11 株式会社バイオサイエンスリンク グリセルアルデヒド３リン酸脱水素酵素（ｇａｐｄｈ）由来ペプチド及びこれを含む抗アレルギー組成物

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US9511124B2 (en) 2016-12-06
EP2705851A4 (en) 2014-10-15
US20140037690A1 (en) 2014-02-06
EP2705851A1 (en) 2014-03-12
EP2705851B1 (en) 2016-07-13
JP2012229164A (ja) 2012-11-22
JP5804592B2 (ja) 2015-11-04

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