WO2014013727A1 - Immunemodulating agent - Google Patents

Immunemodulating agent Download PDF

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WO2014013727A1
WO2014013727A1 PCT/JP2013/004355 JP2013004355W WO2014013727A1 WO 2014013727 A1 WO2014013727 A1 WO 2014013727A1 JP 2013004355 W JP2013004355 W JP 2013004355W WO 2014013727 A1 WO2014013727 A1 WO 2014013727A1
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mangosteen
cells
immunomodulator
suppresses
peel extract
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PCT/JP2013/004355
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French (fr)
Japanese (ja)
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裕明 樋口
安藤　智教
圭司郎 吉田
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株式会社ロッテ
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Priority to JP2014525721A priority Critical patent/JPWO2014013727A1/en
Priority to KR1020157003700A priority patent/KR20150036556A/en
Publication of WO2014013727A1 publication Critical patent/WO2014013727A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • 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
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • 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
    • A61K36/18—Magnoliophyta (angiosperms)
    • 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
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/38—Clusiaceae, Hypericaceae or Guttiferae (Hypericum or Mangosteen family), e.g. common St. Johnswort
    • 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
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients

Definitions

  • the present invention relates to an immunomodulator.
  • An object of the present invention is to provide an immunomodulator that prevents, treats, or reduces immune abnormalities, particularly diseases caused by excessively functioning immune functions.
  • the immune response involves cells such as T cells, B cells, dendritic cells, macrophages, and mast cells.
  • T cells can be divided into helper T cells and killer T cells according to surface antigens.
  • Helper T cells are further divided into Th1 cells and Th2 cells according to cytokine secretion profiles.
  • An immune response in which Th1 cells are dominant is called a Th1-type response and induces cellular immunity
  • an immune response in which Th2 cells are dominant is called a Th2-type response and induces humoral immunity.
  • the Th1 type immune response and the Th2 type immune response are balanced in a normal state, and it is said that if this is greatly biased to either, the disease will be caused. That is, if the Th1 type immune response is enhanced, the autoimmune disease If induced and the Th2-type immune response is enhanced, allergy is said to be induced.
  • Th1 cells When infected with viruses or parasitic bacteria, differentiation of Th0 cells into Th1 cells is promoted, and cellular immunity is activated. Th1 cells produce cytokines such as interferon ⁇ (IFN- ⁇ ) and interleukin (IL) -2, activate T cells and macrophages, and attack virus-infected cells. When the Th1-type immune response is enhanced, self-responsive T cells are activated and may induce chronic inflammation such as rheumatoid arthritis, multiple sclerosis, insulin-dependent diabetes, and autoimmune diseases.
  • IFN- ⁇ interferon ⁇
  • IL interleukin
  • Th0 cells when environmental antigens such as mites and house dust enter the body from the outside, differentiation of Th0 cells into Th2 cells is induced, and humoral immunity is activated.
  • Th2-type response is involved in the development of allergies.
  • environmental antigens such as house dust enter the living body
  • TSLP thymic stromal lymphopoietin
  • TSLP activates dendritic cells and promotes differentiation of Th0 cells into Th2 cells.
  • Activated Th2 cells produce IL-4 and IL-13, which act on B cells, produce antibodies with specificity for the antigen, and induce humoral immunity.
  • IgE antibody may be induced in a large amount by some mechanism.
  • IgE binds to Fc ⁇ RI on mast cells and basophils and crosslinks with the re-invading antigen to promote Fc ⁇ RI aggregation, leading to activation of mast cells and basophils.
  • chemical mediators such as histamine, tryptase, and proteoglycan stored in the cytoplasmic granules are released, causing an immediate allergic reaction.
  • IL-5 produced from Th2 cells induces infiltration of eosinophils into lesions, and chemokines such as CCL22 and CCL24 produced from activated dendritic cells induce induction of Th2 cells into lesions. Inflammatory allergic reactions may be caused.
  • the skin consists of four layers: epidermis, basement membrane, dermis, and subcutaneous tissue.
  • the epidermis is further classified into a horny layer, a granular layer, a spiny layer, and a basal layer.
  • the ordered cell growth and differentiation of keratinocytes which occupy most of the epidermis cell layer, forms a skin barrier, preventing moisture in the stratum corneum from escaping to the outside world, and environmental antigens such as house dust from the outside world in the body. Prevent intrusion.
  • Aquaporin 3 (AQP3: Aquaporin 3) has been reported as a factor that controls cell growth of keratinocytes.
  • AQP3 is a water and glycerin transport protein present on the cell membrane surface, and has been reported to be involved in physiological skin moisturization and wound healing by controlling cell growth / migration and water transport.
  • keratinocytes play an important role in immune responses by releasing inflammatory cytokines and chemokines such as IL-1, IL-6, TNF- ⁇ , TSLP, CCL17 and activating immune cells by various stimuli. Fulfill.
  • NGF nerve growth factor
  • keratinocytes release nerve growth factor (NGF: Nerve growth factor) by various stimuli, increase peripheral nerves and cause hypertrophy, and increase sensitivity to external stimuli.
  • Increased sensitivity to stimuli triggers curettage behavior, which disrupts the epidermal barrier, induces the production of inflammatory cytokines, and creates a vicious circle that further enhances the immune response.
  • Th1 / Th2 cytokines and IgE suppress abnormal growth of keratinocytes and production of inflammatory cytokines and NGF from keratinocytes
  • the substance is expected to be found.
  • Patent Document 1 rice brewed products
  • Patent Document 2 acetic acid and propionic acid
  • the present invention has been made to solve the above-mentioned problems of the prior art, and relates to an immunomodulator having an effect of regulating a safe and immune abnormality consisting of a mangosteen peel extract.
  • Mangosteen (Garcinia mangostana. Lnn) used in the present invention is included in the genus Hypericaceae. Mangosteen is an edible fruit tree that lives in Southeast Asia, and its skin has long been used as a folklore medicine for diarrhea, dysentery, antipyretic drugs and skin diseases. Mangosteen is said to be a fruit queen and is a well-known material with a good image. Therefore, an immunomodulator comprising an extract of mangosteen peel has a good image and is easily accepted by patients.
  • mangosteen extracts particularly ⁇ -mangostin and ⁇ -mangostin, have an inhibitory effect on I ⁇ B kinase (Patent Document 4).
  • mangosteen extract has an effect of preventing / treating atopic dermatitis (Patent Document 5).
  • Non-Patent Document 1 discloses that ⁇ -mangostin suppresses I ⁇ B kinase activity, suppresses the release of prostaglandin E, and consequently suppresses the expression of cyclooxygenase 2 gene.
  • Non-Patent Document 2 discloses that IMD-0354, an inhibitor of I ⁇ B kinase activity, has an effect at the animal level in the treatment of atopic dermatitis, which is one of the immune abnormalities.
  • Non-Patent Document 3 discloses that ⁇ -mangostin has an antiproliferative effect on lymphocytic leukemia B cells.
  • Non-Patent Document 4 discloses that ⁇ -mangosteen and ⁇ -mangostin have an antiproliferative effect on human leukemia cell line HL60.
  • Non-Patent Document 5 discloses that mangosteen peel extract has an antiproliferative effect on human colorectal adenocarcinoma cell line COLO205.
  • mangosteen peel extract extracted from mangosteen peel has a function of regulating immunity, and provides this as an immunomodulator.
  • An immunoregulator comprising a mangosteen peel extract suppresses cell growth and suppresses production of IgE, IFN- ⁇ , TSLP, IL-4, CCL22, CCL24, NGF, AQP3 and the like. Mangosteen has been edible for a long time, and the immunomodulator of the present invention is highly safe.
  • mangosteen peel extract It is the result of investigating the effect of mangosteen peel extract on cell proliferation. It is the result of investigating the effect which a mangosteen peel extract shows with respect to IgE production. It is the result of investigating the effect of mangosteen peel extract on the production of cytokine at the mRNA level. It is the result of investigating the effect of mangosteen peel extract on the production of cytokine at the mRNA level. It is the result of investigating the effect of mangosteen peel extract on the production of TSLP at the mRNA level. It is the result of investigating the effect of mangosteen peel extract on the production of endogenous proteins such as cytokines and chemokines at the mRNA level.
  • the present invention provides an immunomodulator comprising a mangosteen peel extract.
  • An immunomodulator comprising a mangosteen peel extract suppresses cell growth.
  • a cell can mention a keratinocyte, a mast cell, and a B cell as an example.
  • an immunomodulator comprising a mangosteen peel extract suppresses IFN- ⁇ production.
  • the immunomodulator which consists of mangosteen peel extract suppresses the production of TSLP.
  • an immunomodulator comprising mangosteen peel extract suppresses the production of IL-4, CCL22 (MDC), CCL24 (Eotaxin-2), NGF, and AQP3.
  • the present invention also provides an immunomodulator comprising ⁇ -mangostin.
  • An immunomodulatory agent consisting of ⁇ -mangostin suppresses cell proliferation and production of IgE, IFN- ⁇ or TSLP.
  • the present invention also provides an immunomodulator comprising ⁇ -mangostin.
  • An immunomodulator composed of ⁇ -mangostin suppresses cell proliferation and production of IgE or IFN- ⁇ .
  • this invention provides the food / beverage products for immunomodulation containing any of the immunomodulator as described above.
  • Mangosteen is a plant contained in the genus Fuchsia.
  • mangosteen peel those obtained from mangosteen fruit (raw or dried product) can be used.
  • Mangosteen peel extract refers to an extract extracted from mangosteen peel with a solvent.
  • the solvent used for extraction is not particularly limited, but is preferably a polar solvent, and is preferably selected from the group consisting of methanol, ethanol, n-propanol, 2-propanol, n-butanol, acetone, ethyl acetate, and water.
  • the polar solvent to be used or a mixed solvent thereof can be increased. In consideration of use as an orally administered agent or a food or drink, it is preferable in terms of safety to use ethanol or a combination of water and ethanol as the extraction solvent.
  • the extraction temperature is not particularly defined, a range from room temperature to the boiling point of the solvent is preferable in terms of extraction efficiency.
  • the extraction time varies depending on the type of solvent, the state of the skin (raw or dried product, pulverized product, powder, etc.) and the extraction temperature, but a range of 0.5 to 24 hours is preferable.
  • the extract may be concentrated or removed by using an evaporator or the like.
  • the extract can be used after being purified by solvent fractionation or chromatography, if necessary.
  • immunomodulator refers to a regulator that regulates an immune response, and more preferably refers to a regulator that suppresses an excessive immune response in the present invention.
  • immunomodulation include suppression of cell proliferation, suppression of antibody, particularly preferably IgE production, endogenous proteins related to immune responses such as cytokines, chemokines, such as IFN- ⁇ , TSLP, IL-4, Examples include suppression of CCL22, CCL24, NGF, and AQP3.
  • ⁇ -Mangosteen is a compound having the following structure (1) and can be extracted from mangosteen peel.
  • ⁇ -Mangosteen is a compound having the following structure (2) and can be extracted from mangosteen peel.
  • the second fraction (2 g) was purified again by silica gel column chromatography (hexane-ethyl acetate, 30: 70 ⁇ 50: 50, followed by ethyl acetate only, finally ethyl acetate-methanol, 50:50), yellow As an amorphous sample, 500 mg of ⁇ -mangosteen was obtained.
  • the effects of mangosteen peel extract, ⁇ -mangostin and ⁇ -mangostin on the proliferation of keratinocyte cells, mast cells, and B cell cell lines were examined.
  • the mouse keratinocyte cell line PAM212 was cultured in DMEM (Invitrogen, Carlsbad, USA, Cat. No. 12100-046) medium (containing 10% FCS, 100 U / ml PS).
  • Mouse mast cell line P815 was cultured in RPMI 1640 medium (containing 10% FCS, 100 U / ml PS).
  • Mouse B cell line BCL1 was cultured in RPMI 1640 medium (containing 10% FCS, 100 U / ml PS).
  • Mangosteen peel extract, ⁇ -mangosteen and ⁇ -mangostin inhibited the growth of mouse keratinocyte cell line PAM212.
  • the IC50 was 5.9 ⁇ g / ml, 3.8 ⁇ M, and 1.6 ⁇ M, respectively.
  • Mangosteen peel extract, ⁇ -mangosteen and ⁇ -mangostin inhibited the growth of mouse mast cell line P815.
  • the IC50 was 7.7 ⁇ g / ml, 3.0 ⁇ M, and 3.1 ⁇ M, respectively.
  • Mangosteen peel extract, ⁇ -mangosteen and ⁇ -mangostin inhibited the growth of mouse B cell line BCL1.
  • the IC50 was 4.9 ⁇ g / ml, 2.2 ⁇ M, and 1.0 ⁇ M, respectively. Further, the mangosteen peel extract used in this experiment contains 13.2% and 12.4% of ⁇ -mangostin and ⁇ -mangostin, respectively, and the molecular weights are 410.466 and 396.4439, respectively. Considering this, the following formula was used.
  • Contribution ratio of ⁇ -mangosteen ⁇ (IC 50 of mangosteen peel extract) ( ⁇ g / ml) ⁇ 0.131 ⁇ 410.466 ⁇ 1000 ⁇ 10 6 ⁇ ⁇ (IC 50 of ⁇ -mangosteen) ( ⁇ M) ⁇ 100
  • Contribution ratio of ⁇ -mangosteen ⁇ (IC 50 of mangosteen peel extract) ( ⁇ g / ml) ⁇ 0.024 ⁇ 396.439 ⁇ 1000 ⁇ 10 6 ⁇ ⁇ (IC 50 of ⁇ -mangosteen) ( ⁇ M) ⁇ 100
  • Test Method Spleens were isolated from NC / Tnd mice (7-9 weeks old) in an SPF environment, homogenized in RPMI 1640 medium, and a single cell suspension was prepared with a 70 ⁇ m mesh filter. After centrifugation (1200 rpm, 4 ° C., 5 minutes), the suspension was resuspended in Robosp buffer (Stemcell Technologies Inc, Vancouver, Canada, Cat. No. 20104) to a concentration of 1 ⁇ 10 8 cells / ml. Using Mouse CD19 positive selection kit (Stemcell Technologies Inc, Vancouver, Canada, Cat. No. 20000), ROBOSEP (Stemcell Technologies Inc., Candoc.
  • RPMI1640 medium containing 10% FCS, 50 U / ml PS, 1 ⁇ 10 ⁇ 4 M 2-ME
  • 5 ⁇ 10 5 cells / ml It was suspended in. 500 U / ml rmIL-4 (R & D system, Inc., Minneapolis, USA, Cat. No. 404-ML-010) and 10 ⁇ g / ml LPS (Sigma, St. Louis, USA, Cat. No. L6143)
  • Concentration samples were added and seeded in 96-well plates (200 ⁇ l / well).
  • As a control group a group to which no sample was added was provided. After culturing at 37 ° C. under 5% CO 2 for 9 days, the culture supernatant was collected and the IgE concentration was measured by ELISA.
  • Test method Axillary lymph nodes were isolated from NC / Tnd mice (12-17 weeks old) that developed dermatitis in a conventional environment, homogenized in RPMI 1640 medium, and then suspended in a single cell with a 70 ⁇ m mesh filter. A liquid was prepared. After centrifugation (1200 rpm, 4 ° C., 5 min), RPMI1640 medium (10% FCS, 50 U / ml PS, 1 ⁇ 10 ⁇ 4 M 2-ME) to a concentration of 2 ⁇ 10 6 cells / ml Contained).
  • ⁇ CT method a comparative CT method
  • the ⁇ CT method is based on the theory that there is a double amount difference in the expression level due to the difference in detection in one cycle. It is a method for relative quantification of the expression level, paying attention to whether it reaches the Threshold Line.
  • ⁇ CT method a group (medium) not added with PWM was used as a reference.
  • Test Method NC / Tnd mouse neonates (1-3 days) were sacrificed and the skin was cut with scissors. After physically removing the subcutaneous fat, a skin sample was obtained with a 6 mm punch. The skin sample was floated on a 0.25% trypsin-EDTA solution, incubated at 4 ° C. for 1 hour, and then incubated at 37 ° C. for 30 minutes. The epidermis was isolated from the skin sample using tweezers and floated on the medium in a 24-well plate. After adding various concentrations of reagents and pre-incubating at 37 ° C.
  • TNF- ⁇ 25 ng / ml
  • IL-1 ⁇ 10 ng / ml
  • Cultured As a control, a group to which neither a reagent nor TNF- ⁇ and IL-1 ⁇ was added (the leftmost in FIG. 5) and a group to which only TNF- ⁇ and IL-1 ⁇ were added (the second from the left in FIG. 5) were provided.
  • total RNA was extracted using Takara RNA Fastpure, and real-time PCR was performed. Note that ⁇ -actin was used as an endogenous control, and comparison was made by the ⁇ CT method. In the ⁇ CT method, a group to which no reagent, TNF- ⁇ , and IL-1 ⁇ were added was used as a reference.
  • Test method NC / Tnd mice (5-6 weeks old) who have not yet developed dermatitis in a conventional environment, a control diet administration group, a mangosteen administration group which is an administration group of a mixed diet containing mangosteen peel extract, Into two groups (each group n 7). All groups were allowed to freely ingest each test feed after a one-week preliminary breeding using a control feed in a conventional environment. That is, the mangosteen administration group was given a mixed diet containing 0.25% of mangosteen peel extract (the intake of mangosteen peel extract was 250 mg / kg / day), and the control sample administration group had CRF- 1 (Oriental Yeast) was administered.
  • mice After administration of the test feed for 6 weeks, the mice were sacrificed by cervical dislocation and skin samples were obtained by 6 mm punch. From the obtained skin sample, total RNA was isolated using TAKARA FastPure RNA kit (TAKARA Bio Inc., Shiga, Japan, Cat. No. 9190), and TAKARA PrimeScript 1st strand cDNA Synthesis Science. , Japan, Cat.No. 6110A), and mRNA expression of various cytokines, chemokines, NGF and semaphorin 3a (Sema3a) involved in the regulation of nerve fiber extension, and AQP3, a cell growth regulator. Real-time PCR was performed to analyze the quantity. Note that ⁇ -actin was used as an endogenous control, and comparison was made by the ⁇ CT method. In the ⁇ CT method, the control feed group was used as a reference.
  • TAKARA FastPure RNA kit TAKARA Bio Inc., Shiga, Japan, Cat. No. 9190
  • the vertical axis is a relative value where the value in the control feed group is 1.
  • the expression levels of TSLP, IL-4, IFN- ⁇ , CCL22 (MDC), CCL24 (Eotaxin-2), NGF, and AQP3 mRNA in the skin were significantly low.

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Abstract

Provided is an immunomodulating agent for modulating abnormal immunity, particularly an excess immune response. The present inventors have made extensive studies. As a result, it is found that a mangosteen peel extract produced by extracting a mangosteen peel with a polar solvent has a function of modulating immunity, and the mangosteen peel extract is provided as an immunomodulating agent.

Description

免疫調節剤Immunomodulator
　本発明は免疫調節剤に関する。 The present invention relates to an immunomodulator.
　高等動物は、細菌、ウイルス、毒物などの異物が侵入してきた際に体を防御する、免疫機能を備えている。免疫機能が低下すると、細菌などによる感染症にかかりやすくなる。一方、免疫機能が過剰に働くとアレルギーや自己免疫疾患といった疾病を招く。本発明では、免疫異常、特に過剰に働く免疫機能によって引き起こされる疾病を予防、治療、あるいは軽減する免疫調節剤の提供を目的とする。 Higher animals have an immune function that protects the body when foreign bodies such as bacteria, viruses, and poisons enter. If the immune function is reduced, infection by bacteria is likely to occur. On the other hand, excessive immune function leads to diseases such as allergies and autoimmune diseases. An object of the present invention is to provide an immunomodulator that prevents, treats, or reduces immune abnormalities, particularly diseases caused by excessively functioning immune functions.
　免疫応答には、Ｔ細胞、Ｂ細胞、樹状細胞、マクロファージ、肥満細胞などの細胞が関与している。Ｔ細胞は、表面抗原により、ヘルパーＴ細胞、キラーＴ細胞に分けることができ、ヘルパーＴ細胞はさらにサイトカインの分泌のプロファイルによりＴｈ１細胞とＴｈ２細胞に分けられる。Ｔｈ１細胞が優勢な免疫応答はＴｈ１型応答といわれ細胞性免疫を誘導し、Ｔｈ２細胞が優勢な免疫応答はＴｈ２型応答といわれ液性免疫を誘導する。Ｔｈ１型免疫応答とＴｈ２型免疫応答は正常な状態ではバランスが保たれており、これがどちらかに大きく偏ると疾患を起こすといわれ、すなわち、Ｔｈ１型の免疫応答が亢進すれば、自己免疫疾患が誘導され、Ｔｈ２型の免疫応答が亢進すればアレルギーが誘導されるといわれる。 The immune response involves cells such as T cells, B cells, dendritic cells, macrophages, and mast cells. T cells can be divided into helper T cells and killer T cells according to surface antigens. Helper T cells are further divided into Th1 cells and Th2 cells according to cytokine secretion profiles. An immune response in which Th1 cells are dominant is called a Th1-type response and induces cellular immunity, and an immune response in which Th2 cells are dominant is called a Th2-type response and induces humoral immunity. The Th1 type immune response and the Th2 type immune response are balanced in a normal state, and it is said that if this is greatly biased to either, the disease will be caused. That is, if the Th1 type immune response is enhanced, the autoimmune disease If induced and the Th2-type immune response is enhanced, allergy is said to be induced.
　ウイルス、寄生菌に感染すると、Ｔｈ０細胞のＴｈ１細胞への分化が促され、細胞性免疫が活発化される。Ｔｈ１細胞はインターフェロンγ（ＩＦＮ－γ）やインターロイキン（ＩＬ）－２等のサイトカインを産生し、Ｔ細胞、マクロファージなどを活発化し、ウイルス感染した細胞を攻撃する。Ｔｈ１型免疫応答が亢進すると自己応答型のＴ細胞を活性化し、関節リウマチや多発性硬化症、インスリン依存性糖尿病等の慢性炎症や自己免疫疾患を誘導する場合がある。 When infected with viruses or parasitic bacteria, differentiation of Th0 cells into Th1 cells is promoted, and cellular immunity is activated. Th1 cells produce cytokines such as interferon γ (IFN-γ) and interleukin (IL) -2, activate T cells and macrophages, and attack virus-infected cells. When the Th1-type immune response is enhanced, self-responsive T cells are activated and may induce chronic inflammation such as rheumatoid arthritis, multiple sclerosis, insulin-dependent diabetes, and autoimmune diseases.
　一方、外界からダニやハウスダストなどの環境抗原が体内に侵入するとＴｈ０細胞のＴｈ２細胞への分化が誘導され、液性免疫が活発化される。 On the other hand, when environmental antigens such as mites and house dust enter the body from the outside, differentiation of Th0 cells into Th2 cells is induced, and humoral immunity is activated.
　アレルギーの発症にはＴｈ２型応答が関わっている。ハウスダストなどの環境抗原が生体に侵入すると、表皮ケラチノサイトより胸腺間質性リンパ球新生因子（ＴＳＬＰ：ｔｈｙｍｉｃ　ｓｔｒｏｍａｌ　ｌｙｍｐｈｏｐｏｉｅｔｉｎ）が産生される。ＴＳＬＰは樹状細胞を活性化し、Ｔｈ０細胞のＴｈ２細胞への分化を促す。活性化したＴｈ２細胞からＩＬ－４、ＩＬ－１３が産生され、これらがＢ細胞に作用し、抗原に対して特異性を有する抗体が産生され、液性免疫が誘導される。この際に、なんらかの機序によりＩｇＥ抗体が多量に誘導される場合がある。ＩｇＥは肥満細胞及び好塩基球上のＦｃεＲＩと結合し、再度侵入した抗原と架橋すると、ＦｃεＲＩの凝集が促進され、肥満細胞や好塩基球の活性化が引き起こされる。その際に、細胞質顆粒内に蓄えられたヒスタミン、トリプターゼ、プロテオグリカンといった化学伝達物質が放出され、即時性アレルギー反応が引き起こされる。更に、Ｔｈ２細胞より産生されるＩＬ－５により好酸球の病変部への浸潤が、また活性化樹状細胞より産生されるＣＣＬ２２やＣＣＬ２４といったケモカインによりＴｈ２細胞の病変部への浸潤が誘導され、炎症アレルギー反応が引き起こされる場合がある。 Ｈ Th2-type response is involved in the development of allergies. When environmental antigens such as house dust enter the living body, thymic stromal lymphopoietin (TSLP) is produced from epidermal keratinocytes. TSLP activates dendritic cells and promotes differentiation of Th0 cells into Th2 cells. Activated Th2 cells produce IL-4 and IL-13, which act on B cells, produce antibodies with specificity for the antigen, and induce humoral immunity. At this time, IgE antibody may be induced in a large amount by some mechanism. IgE binds to FcεRI on mast cells and basophils and crosslinks with the re-invading antigen to promote FcεRI aggregation, leading to activation of mast cells and basophils. At that time, chemical mediators such as histamine, tryptase, and proteoglycan stored in the cytoplasmic granules are released, causing an immediate allergic reaction. Furthermore, IL-5 produced from Th2 cells induces infiltration of eosinophils into lesions, and chemokines such as CCL22 and CCL24 produced from activated dendritic cells induce induction of Th2 cells into lesions. Inflammatory allergic reactions may be caused.
　近年、アレルギーには、皮膚のバリア機能の破綻も関与すると考えられはじめている。
　皮膚は表皮、基底膜、真皮、皮下組織の４層からなり、表皮は更に角層、顆粒層、有棘層、基底層に分類される。表皮細胞層の大部分を占めるケラチノサイトの秩序だった細胞増殖・分化により、皮膚バリアが形成され、角層内水分が外界に逃げるのを防ぐとともに、外界からのハウスダストなどの環境抗原が体内に侵入するのを防ぐ。乾癬、光線性角化症、魚鱗癬、脂漏性皮膚炎等の皮膚疾患では、ケラチノサイトの細胞増殖制御が破綻をきたし、皮膚上皮細胞の異常増殖により皮膚の肥厚化が起こる。その結果、表皮バリア機能が低下することで、環境抗原が侵入しやすくなる。 In recent years, allergies are beginning to be considered to involve the breakdown of the barrier function of the skin.
The skin consists of four layers: epidermis, basement membrane, dermis, and subcutaneous tissue. The epidermis is further classified into a horny layer, a granular layer, a spiny layer, and a basal layer. The ordered cell growth and differentiation of keratinocytes, which occupy most of the epidermis cell layer, forms a skin barrier, preventing moisture in the stratum corneum from escaping to the outside world, and environmental antigens such as house dust from the outside world in the body. Prevent intrusion. In skin diseases such as psoriasis, actinic keratosis, ichthyosis, and seborrheic dermatitis, cell proliferation control of keratinocytes is broken, and skin thickening occurs due to abnormal proliferation of skin epithelial cells. As a result, the epidermal barrier function is lowered, so that environmental antigens can easily enter.
　ケラチノサイトの細胞増殖を制御する因子として、アクアポリン３（ＡＱＰ３：Ａｑｕａｐｏｒｉｎ３）が報告されている。ＡＱＰ３は、細胞膜表面に存在する水及びグリセリンの輸送タンパク質であり、細胞の増殖・移動と水分の輸送を制御し、皮膚の生理的保湿や創傷治癒に関与することが報告されている。また、ケラチノサイトは種々の刺激により、ＩＬ－１、ＩＬ－６、ＴＮＦ－α、ＴＳＬＰ、ＣＣＬ１７等の炎症性サイトカインやケモカインを放出し、免疫細胞を活性化させるなど免疫応答においても重要な役割を果たす。更に、ケラチノサイトは種々の刺激により、神経成長因子（ＮＧＦ：Ｎｅｒｖｅ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ）を放出し、末梢神経の増加・肥大を引き起こし、外界の刺激に対する感受性が増大することが報告されている。刺激に対する感受性が増大すると、掻爬行動が惹起され、すると、表皮バリアが破壊され、炎症性サイトカインの産生が誘導され、免疫応答がさらに亢進するという悪循環となる。 Aquaporin 3 (AQP3: Aquaporin 3) has been reported as a factor that controls cell growth of keratinocytes. AQP3 is a water and glycerin transport protein present on the cell membrane surface, and has been reported to be involved in physiological skin moisturization and wound healing by controlling cell growth / migration and water transport. In addition, keratinocytes play an important role in immune responses by releasing inflammatory cytokines and chemokines such as IL-1, IL-6, TNF-α, TSLP, CCL17 and activating immune cells by various stimuli. Fulfill. Furthermore, it has been reported that keratinocytes release nerve growth factor (NGF: Nerve growth factor) by various stimuli, increase peripheral nerves and cause hypertrophy, and increase sensitivity to external stimuli. Increased sensitivity to stimuli triggers curettage behavior, which disrupts the epidermal barrier, induces the production of inflammatory cytokines, and creates a vicious circle that further enhances the immune response.
　免疫疾患の予防・治療には、免疫機能と表皮バリア機能を上手く制御する物質、即ちＴｈ１／Ｔｈ２サイトカイン、ＩｇＥ産生に加え、ケラチノサイトの異常増殖やケラチノサイトからの炎症性サイトカインやＮＧＦの産生を抑制する物質が見出されることが期待される。 For prevention and treatment of immune diseases, in addition to production of substances that successfully control immune function and epidermal barrier function, ie, Th1 / Th2 cytokines and IgE, suppress abnormal growth of keratinocytes and production of inflammatory cytokines and NGF from keratinocytes The substance is expected to be found.
　このような背景の下、免疫調節能を有する物の探索が行われており、米原料醸造物（特許文献１）・酢酸及びプロピオン酸（特許文献２）等が開示されている。しかしこれらは、いずれもＴｈ１／Ｔｈ２バランスに対する効果を有するのみで、表皮バリア改善も含む多面的効果を有することは開示されていない。 Under such a background, search for products having immunomodulating ability has been conducted, and rice brewed products (Patent Document 1), acetic acid and propionic acid (Patent Document 2), etc. are disclosed. However, these have only an effect on the Th1 / Th2 balance, and are not disclosed to have a multifaceted effect including improvement of the epidermal barrier.
　本発明は、上記のような従来技術の問題点を解決するためになされたもので、マンゴスチン果皮抽出物中からなる安全かつ免疫異常を調節する効果を有する免疫調節剤に関するものである。 The present invention has been made to solve the above-mentioned problems of the prior art, and relates to an immunomodulator having an effect of regulating a safe and immune abnormality consisting of a mangosteen peel extract.
　本発明で使用するマンゴスチン（Ｇａｒｃｉｎｉａ　ｍａｎｇｏｓｔａｎａ．　Ｌｎｎ）はオトギリソウ科フクギ属に含まれる。マンゴスチンは東南アジアに生息する食用果樹であり、その果皮は古くから民間伝承薬として下痢、赤痢、解熱剤や皮膚病に利用されてきた。マンゴスチンはフルーツの女王といわれ、知名度も高くイメージのよい素材である。したがってマンゴスチン果皮の抽出物からなる免疫調整剤は、イメージがよく、患者に受け入れられやすい。 Mangosteen (Garcinia mangostana. Lnn) used in the present invention is included in the genus Hypericaceae. Mangosteen is an edible fruit tree that lives in Southeast Asia, and its skin has long been used as a folklore medicine for diarrhea, dysentery, antipyretic drugs and skin diseases. Mangosteen is said to be a fruit queen and is a well-known material with a good image. Therefore, an immunomodulator comprising an extract of mangosteen peel has a good image and is easily accepted by patients.
　マンゴスチンの効能については、マンゴスチンの抽出物から精製されたα－マンゴスチン及びγ－マンゴスチンが、抗ヒスタミン効果あるいは抗セロトニン効果を有すること、また、α－マンゴスチン及びγ－マンゴスチンがヒト花粉症に対する抑制効果を示すことが知られている（特許文献３）。また、マンゴスチン抽出物、とりわけα－マンゴスチン、γ－マンゴスチンがＩκＢキナーゼに対する阻害効果を有することも知られている（特許文献４）。さらに、マンゴスチン抽出物がアトピー性皮膚炎を予防／治療する効果を有することも知られている（特許文献５）。 Regarding the efficacy of mangosteen, α-mangostin and γ-mangosteen purified from mangosteen extract have an antihistamine effect or an anti-serotonin effect, and α-mangostin and γ-mangostin have an inhibitory effect on human pollinosis. (Patent Document 3). It is also known that mangosteen extracts, particularly α-mangostin and γ-mangostin, have an inhibitory effect on IκB kinase (Patent Document 4). Furthermore, it is also known that mangosteen extract has an effect of preventing / treating atopic dermatitis (Patent Document 5).
　また、非特許文献１はγ－マンゴスチンがＩκＢキナーゼ活性を抑制すること、プロスタグランジンＥの放出を抑制し、その結果シクロオキシゲナーゼ２遺伝子の発現を抑制することを開示する。非特許文献２は、ＩκＢキナーゼ活性阻害剤であるＩＭＤ―０３５４が免疫異常の一つであるアトピー性皮膚炎の治療に動物レベルで効果を有することを開示する。非特許文献３はα－マンゴスチンがリンパ性白血病Ｂ細胞に対して、抗増殖効果を有することを開示する。非特許文献４はα－マンゴスチン及びγ－マンゴスチンがヒト白血病細胞株ＨＬ６０に対し抗増殖効果を有することを開示する。 Non-Patent Document 1 discloses that γ-mangostin suppresses IκB kinase activity, suppresses the release of prostaglandin E, and consequently suppresses the expression of cyclooxygenase 2 gene. Non-Patent Document 2 discloses that IMD-0354, an inhibitor of IκB kinase activity, has an effect at the animal level in the treatment of atopic dermatitis, which is one of the immune abnormalities. Non-Patent Document 3 discloses that α-mangostin has an antiproliferative effect on lymphocytic leukemia B cells. Non-Patent Document 4 discloses that α-mangosteen and γ-mangostin have an antiproliferative effect on human leukemia cell line HL60.
　非特許文献５はマンゴスチン果皮抽出物がヒト結腸直腸腺癌の細胞株ＣＯＬＯ２０５に対し抗増殖効果を有することを開示する。 Non-Patent Document 5 discloses that mangosteen peel extract has an antiproliferative effect on human colorectal adenocarcinoma cell line COLO205.
特開２００８－１９５７１３JP2008-195713 特開２００９－２９２７８５JP2009-292785 特許第３９６８４０５Patent No. 3968405 特開２０１０－１９５８３１JP 2010-195831 A 再表２００９－１４２３２０Table 2009-142320
　免疫異常、特に過剰な免疫応答を調節するための免疫調節剤の提供。 Provision of immunomodulators for regulating immune abnormalities, especially excessive immune responses.
　発明者らは鋭意研究の結果マンゴスチンの果皮から抽出したマンゴスチン果皮抽出物が免疫を調節する機能を有することを見出し、これを免疫調節剤として提供する。 As a result of intensive studies, the inventors have found that a mangosteen peel extract extracted from mangosteen peel has a function of regulating immunity, and provides this as an immunomodulator.
　マンゴスチン果皮抽出物からなる免疫調節剤は、細胞の増殖の抑制、ＩｇＥ、ＩＦＮ－γ、ＴＳＬＰ、ＩＬ－４、ＣＣＬ２２、ＣＣＬ２４、ＮＧＦ、ＡＱＰ３等の産生を抑制する。
　マンゴスチンは、古くから食用されており、本発明の免疫調節剤は安全性が高い。 An immunoregulator comprising a mangosteen peel extract suppresses cell growth and suppresses production of IgE, IFN-γ, TSLP, IL-4, CCL22, CCL24, NGF, AQP3 and the like.
Mangosteen has been edible for a long time, and the immunomodulator of the present invention is highly safe.
細胞の増殖に対してマンゴスチン果皮抽出物が示す効果を調べた結果である。It is the result of investigating the effect of mangosteen peel extract on cell proliferation. ＩｇＥ産生に対してマンゴスチン果皮抽出物が示す効果を調べた結果である。It is the result of investigating the effect which a mangosteen peel extract shows with respect to IgE production. サイトカインの産生に対してマンゴスチン果皮抽出物が示す効果をｍＲＮＡレベルで調べた結果である。It is the result of investigating the effect of mangosteen peel extract on the production of cytokine at the mRNA level. サイトカインの産生に対してマンゴスチン果皮抽出物が示す効果をｍＲＮＡレベルで調べた結果である。It is the result of investigating the effect of mangosteen peel extract on the production of cytokine at the mRNA level. ＴＳＬＰの産生に対してマンゴスチン果皮抽出物が示す効果をｍＲＮＡレベルで調べた結果である。It is the result of investigating the effect of mangosteen peel extract on the production of TSLP at the mRNA level. サイトカイン、ケモカインなどの内因性タンパク質の産生に対してマンゴスチン果皮抽出物が示す効果をｍＲＮＡレベルで調べた結果である。It is the result of investigating the effect of mangosteen peel extract on the production of endogenous proteins such as cytokines and chemokines at the mRNA level.
　本発明は、マンゴスチン果皮抽出物からなる免疫調節剤を提供する。マンゴスチン果皮抽出物からなる免疫調節剤は、細胞の増殖を抑制する。細胞の種類は限定されないが、細胞はケラチノサイト、肥満細胞及びＢ細胞を例としてあげることができる。また、マンゴスチン果皮抽出物からなる免疫調節剤は、ＩＦＮ－γの産生を抑制する。また、マンゴスチン果皮抽出物からなる免疫調節剤はＴＳＬＰの産生を抑制する。さらには、マンゴスチン果皮抽出物からなる免疫調節剤はＩＬ－４、ＣＣＬ２２（ＭＤＣ）、ＣＣＬ２４（Ｅｏｔａｘｉｎ－２）、ＮＧＦ、ＡＱＰ３の産生を抑制する。 The present invention provides an immunomodulator comprising a mangosteen peel extract. An immunomodulator comprising a mangosteen peel extract suppresses cell growth. Although the kind of cell is not limited, a cell can mention a keratinocyte, a mast cell, and a B cell as an example. In addition, an immunomodulator comprising a mangosteen peel extract suppresses IFN-γ production. Moreover, the immunomodulator which consists of mangosteen peel extract suppresses the production of TSLP. Furthermore, an immunomodulator comprising mangosteen peel extract suppresses the production of IL-4, CCL22 (MDC), CCL24 (Eotaxin-2), NGF, and AQP3.
　また、本発明は、α－マンゴスチンからなる免疫調節剤を提供する。α－マンゴスチンからなる免疫調節剤は細胞の増殖、また、ＩｇＥ、ＩＦＮ－γまたはＴＳＬＰの産生を抑制する。 The present invention also provides an immunomodulator comprising α-mangostin. An immunomodulatory agent consisting of α-mangostin suppresses cell proliferation and production of IgE, IFN-γ or TSLP.
　また、本発明は、γ－マンゴスチンからなる免疫調節剤を提供する。γ－マンゴスチンからなる免疫調節剤は細胞の増殖、また、ＩｇＥ、またはＩＦＮ－γの産生を抑制する。
　さらに本発明は上記に記載の免疫調節剤のいずれかを含む、免疫調節のための飲食品を提供する。 The present invention also provides an immunomodulator comprising γ-mangostin. An immunomodulator composed of γ-mangostin suppresses cell proliferation and production of IgE or IFN-γ.
Furthermore, this invention provides the food / beverage products for immunomodulation containing any of the immunomodulator as described above.
　マンゴスチンはオトギリソウ科フクギ属に含まれる植物である。マンゴスチンの果皮は、マンゴスチン果実（生または乾燥品）から得られるものを使用することができる。 Mangosteen is a plant contained in the genus Fuchsia. As the mangosteen peel, those obtained from mangosteen fruit (raw or dried product) can be used.
　マンゴスチン果皮抽出物とは、マンゴスチン果皮から溶媒により抽出された抽出物をいう。抽出に用いられる溶媒は、特に限定されないが、好ましくは極性溶媒であり、好ましい例としてメタノール、エタノール、ｎ－プロパノール、２－プロパノール、ｎ―ブタノール、アセトン、酢酸エチル、および水からなる群から選択される極性溶媒あるいはそれらの混合溶媒を上げることができる。経口投与剤又は飲食品として用いることを考慮すると、抽出溶剤として、エタノール、又は水とエタノールとの組み合わせを用いることが安全性の点において好ましい。抽出の温度は特に規定はしないが、抽出効率の点で、室温から溶媒の沸点温度の範囲が好適である。抽出時間は溶媒の種類、果皮の状態（生または乾燥品、粉砕物または粉末など）および抽出温度により変化するが、０．５～２４時間の範囲が好適である。 “Mangosteen peel extract” refers to an extract extracted from mangosteen peel with a solvent. The solvent used for extraction is not particularly limited, but is preferably a polar solvent, and is preferably selected from the group consisting of methanol, ethanol, n-propanol, 2-propanol, n-butanol, acetone, ethyl acetate, and water. The polar solvent to be used or a mixed solvent thereof can be increased. In consideration of use as an orally administered agent or a food or drink, it is preferable in terms of safety to use ethanol or a combination of water and ethanol as the extraction solvent. Although the extraction temperature is not particularly defined, a range from room temperature to the boiling point of the solvent is preferable in terms of extraction efficiency. The extraction time varies depending on the type of solvent, the state of the skin (raw or dried product, pulverized product, powder, etc.) and the extraction temperature, but a range of 0.5 to 24 hours is preferable.
　抽出物は必要によりエバポレーターなどにより抽出溶剤を濃縮し、あるいは除去してもよい。また、抽出物は、必要により溶媒分画やクロマトグラフィーにより精製して用いることもできる。 If necessary, the extract may be concentrated or removed by using an evaporator or the like. In addition, the extract can be used after being purified by solvent fractionation or chromatography, if necessary.
　免疫調節剤とは、免疫応答を調節する調節剤を指し、本発明においてはより好ましくは、過剰な免疫応答を抑制する調節剤を指す。免疫調節の例としては、細胞の増殖の抑制、抗体、特に好ましくはＩｇＥの産生の抑制、サイトカイン、ケモカインといった、免疫応答に関連する内因性タンパク質、たとえば、ＩＦＮ－γ、ＴＳＬＰ、ＩＬ－４，ＣＣＬ２２，ＣＣＬ２４、ＮＧＦ、ＡＱＰ３の抑制を挙げることができる。 The term “immunomodulator” refers to a regulator that regulates an immune response, and more preferably refers to a regulator that suppresses an excessive immune response in the present invention. Examples of immunomodulation include suppression of cell proliferation, suppression of antibody, particularly preferably IgE production, endogenous proteins related to immune responses such as cytokines, chemokines, such as IFN-γ, TSLP, IL-4, Examples include suppression of CCL22, CCL24, NGF, and AQP3.
　α－マンゴスチンとは、次の（１）の構造をもつ化合物であり、マンゴスチン果皮より抽出が可能である。
Figure JPOXMLDOC01-appb-C000001
 α-Mangosteen is a compound having the following structure (1) and can be extracted from mangosteen peel.
Figure JPOXMLDOC01-appb-C000001
　γ－マンゴスチンとは、次の（２）の構造をもつ化合物であり、マンゴスチン果皮より抽出が可能である。
Figure JPOXMLDOC01-appb-C000002
 γ-Mangosteen is a compound having the following structure (2) and can be extracted from mangosteen peel.
Figure JPOXMLDOC01-appb-C000002
・マンゴスチン果皮抽出物の調製
　マンゴスチンの未乾燥果皮１００ｇを粉砕し、１ｌの７０％エタノール中で１時間、８０℃で撹拌抽出した。これをろ過し、ろ液をエバポレーターで減圧乾燥し、２７．４ｇの抽出物を得た。これをマンゴスチン果皮抽出物とした。以下、本明細書中において、この抽出物をＭＥ７０と呼称することがある。 -Preparation of mangosteen peel extract 100 g of undried mangosteen peel was pulverized and extracted with stirring in 1 l of 70% ethanol for 1 hour at 80 ° C. This was filtered, and the filtrate was dried under reduced pressure with an evaporator to obtain 27.4 g of an extract. This was used as a mangosteen peel extract. Hereinafter, this extract may be referred to as ME70 in the present specification.
・α－マンゴスチン、γ－マンゴスチンの調製
　マンゴスチンの未乾燥果皮１ｋｇを１０Ｌのメタノールに浸漬し、２４時間室温下抽出した。濾過後、濾液をエバポレーターで減圧乾燥し、８０ｇの抽出物を得た。得られた抽出物８０ｇを３５０ｍｌの酢酸エチルに溶解後、２００ｍｌの水で２回洗浄した。酢酸エチル画分をエバポレーターで溶媒を溜去させ２０ｇの乾燥物を得た。この乾燥物をシリカゲルカラムクロマトグラフィーで精製した。溶出はヘキサン－酢酸エチル系で漸次、極性をあげるグラジエント溶出を行い、３つの画分を得た。最初に得られた画分（５ｇ）を再度、シリカゲルカラムクロマトグラフィー（ヘキサン－酢酸エチル、１０：９０→３０：７０→５０：５０）で精製し、黄色結晶状の試料としてα－マンゴスチン２ｇを得た。２つめの画分（２ｇ）を再度シリカゲルカラムクロマトグラフィー（ヘキサン－酢酸エチル、３０：７０→５０：５０、続いて酢酸エチルのみ、最後に酢酸エチル－メタノール、５０：５０）で精製し、黄色非結晶状の試料としてγ－マンゴスチン５００ｍｇを得た。 Preparation of α-mangosteen and γ-mangostin 1 kg of undried mangosteen peel was immersed in 10 L of methanol and extracted at room temperature for 24 hours. After filtration, the filtrate was dried under reduced pressure with an evaporator to obtain 80 g of an extract. 80 g of the obtained extract was dissolved in 350 ml of ethyl acetate and then washed twice with 200 ml of water. The ethyl acetate fraction was evaporated using an evaporator to obtain 20 g of a dried product. This dried product was purified by silica gel column chromatography. Elution was performed in a hexane-ethyl acetate system, and gradient elution was performed to gradually increase the polarity, and three fractions were obtained. The first fraction (5 g) obtained was purified again by silica gel column chromatography (hexane-ethyl acetate, 10: 90 → 30: 70 → 50: 50), and 2 g of α-mangosteen was obtained as a yellow crystalline sample. Obtained. The second fraction (2 g) was purified again by silica gel column chromatography (hexane-ethyl acetate, 30: 70 → 50: 50, followed by ethyl acetate only, finally ethyl acetate-methanol, 50:50), yellow As an amorphous sample, 500 mg of γ-mangosteen was obtained.
・試験法
　ケラチノサイト細胞、肥満細胞、及びＢ細胞の細胞株の増殖に対してマンゴスチン果皮抽出物、α－マンゴスチン、γ－マンゴスチンが示す効果を調べた。
　マウスケラチノサイト細胞株ＰＡＭ２１２はＤＭＥＭ（Ｉｎｖｉｔｒｏｇｅｎ，Ｃａｒｌｓｂａｄ，ＵＳＡ，Ｃａｔ．Ｎｏ．１２１００－０４６）培地（１０％ＦＣＳ、１００Ｕ／ｍｌ　Ｐ－Ｓ含有）で培養した。マウス肥満細胞株Ｐ８１５は　ＲＰＭＩ１６４０培地（１０％　ＦＣＳ、１００Ｕ／ｍｌ　Ｐ－Ｓ含有）で培養した。マウスＢ細胞株ＢＣＬ１は　ＲＰＭＩ１６４０培地（１０％　ＦＣＳ、１００Ｕ／ｍｌ　Ｐ－Ｓ含有）で培養した。
　各細胞の懸濁液に様々な濃度の試料（マンゴスチン果皮抽出物（ＭＥ７０）、α－マンゴスチン、γ－マンゴスチン、ＩＭＤ－０３５４，ＦＫ５０６）を加え、９６ウェルプレートに播種した（１００μｌ／ｗｅｌｌ）。対照群として、試料を添加しない群を設けた。ＦＫ５０６は公知の免疫抑制剤である。
　なお、ＰＡＭ２１２は２×１０４細胞／ｍｌの濃度で播種を行った。Ｐ８１５は５×１０４細胞／ｍｌの濃度で播種を行った。ＢＣＬ１は２×１０５細胞／ｍｌの濃度で播種を行った。
　播種２４、４８、７２時間後にｔｒｙｐａｎ　ｂｌｕｅ　ｄｙｅ　ｅｘｃｌｕｓｉｏｎ　ｔｅｓｔにより生細胞数の測定を行った。 Test Method The effects of mangosteen peel extract, α-mangostin and γ-mangostin on the proliferation of keratinocyte cells, mast cells, and B cell cell lines were examined.
The mouse keratinocyte cell line PAM212 was cultured in DMEM (Invitrogen, Carlsbad, USA, Cat. No. 12100-046) medium (containing 10% FCS, 100 U / ml PS). Mouse mast cell line P815 was cultured in RPMI 1640 medium (containing 10% FCS, 100 U / ml PS). Mouse B cell line BCL1 was cultured in RPMI 1640 medium (containing 10% FCS, 100 U / ml PS).
Various concentrations of samples (mangosteen peel extract (ME70), α-mangosteen, γ-mangosteen, IMD-0354, FK506) were added to each cell suspension and seeded in a 96-well plate (100 μl / well). As a control group, a group to which no sample was added was provided. FK506 is a known immunosuppressive agent.
PAM212 was seeded at a concentration of 2 × 10 4 cells / ml. P815 was seeded at a concentration of 5 × 10 4 cells / ml. BCL1 was seeded at a concentration of 2 × 10 5 cells / ml.
Viable cell count was measured by trypan blue dye exclusion test 24, 48, and 72 hours after seeding.
・結果
　結果を表１および図１に示す。マンゴスチン果皮抽出物、α－マンゴスチン、γ－マンゴスチンはマウスケラチノサイト細胞株ＰＡＭ２１２の増殖を抑制した。ＩＣ５０はそれぞれ５．９μｇ／ｍｌ、３．８μＭ、１．６μＭであった。
　マンゴスチン果皮抽出物、α－マンゴスチン、γ－マンゴスチンはマウス肥満細胞株Ｐ８１５の増殖を抑制した。ＩＣ５０はそれぞれ７．７μｇ／ｍｌ、３．０μＭ、３．１μＭであった。
　マンゴスチン果皮抽出物、α－マンゴスチン、γ－マンゴスチンはマウスＢ細胞株ＢＣＬ１の増殖を抑制した。ＩＣ５０はそれぞれ４．９μｇ／ｍｌ、２．２μＭ、１．０μＭであった。
　また、本実験で使用したマンゴスチン果皮抽出物には、α―マンゴスチン、γ―マンゴスチンがそれぞれ１３．２％、１２．４％含まれ、分子量が、それぞれ４１０．４６６、３９６．４３９であることを考慮し、以下の式により求めた。
α－マンゴスチンの寄与率＝｛（マンゴスチン果皮抽出物のＩＣ５０）（μｇ／ｍｌ）×０．１３１÷４１０．４６６÷１０００×１０６｝÷（α－マンゴスチンのＩＣ５０）（μＭ）×１００
γ－マンゴスチンの寄与率＝｛（マンゴスチン果皮抽出物のＩＣ５０）（μｇ／ｍｌ）×０．０２４÷３９６．４３９÷１０００×１０６｝÷（γ－マンゴスチンのＩＣ５０）（μＭ）×１００ -Results The results are shown in Table 1 and FIG. Mangosteen peel extract, α-mangosteen and γ-mangostin inhibited the growth of mouse keratinocyte cell line PAM212. The IC50 was 5.9 μg / ml, 3.8 μM, and 1.6 μM, respectively.
Mangosteen peel extract, α-mangosteen and γ-mangostin inhibited the growth of mouse mast cell line P815. The IC50 was 7.7 μg / ml, 3.0 μM, and 3.1 μM, respectively.
Mangosteen peel extract, α-mangosteen and γ-mangostin inhibited the growth of mouse B cell line BCL1. The IC50 was 4.9 μg / ml, 2.2 μM, and 1.0 μM, respectively.
Further, the mangosteen peel extract used in this experiment contains 13.2% and 12.4% of α-mangostin and γ-mangostin, respectively, and the molecular weights are 410.466 and 396.4439, respectively. Considering this, the following formula was used.
Contribution ratio of α-mangosteen = {(IC 50 of mangosteen peel extract) (μg / ml) × 0.131 ÷ 410.466 ÷ 1000 × 10 6 } ÷ (IC 50 of α-mangosteen) (μM) × 100
Contribution ratio of γ-mangosteen = {(IC 50 of mangosteen peel extract) (μg / ml) × 0.024 ÷ 396.439 ÷ 1000 × 10 6 } ÷ (IC 50 of γ-mangosteen) (μM) × 100
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
・試験法
　ＳＰＦ環境下のＮＣ／Ｔｎｄマウス（７～９週齢）から、脾臓を単離し、ＲＰＭＩ１６４０培地中でホモジェナイズ後、７０μｍメッシュフィルターにより、単一細胞懸濁液を作製した。遠心分離後（１２００ｒｐｍ、４℃、５分）、１×１０８細胞／ｍｌの濃度になるように、Ｒｏｂｏｓｅｐバッファー（Ｓｔｅｍｃｅｌｌ　Ｔｅｃｈｎｏｌｏｇｉｅｓ　Ｉｎｃ，　Ｖａｎｃｏｕｖｅｒ，Ｃａｎａｄａ，Ｃａｔ．Ｎｏ．２０１０４）に再懸濁した。Ｍｏｕｓｅ　ＣＤ１９　ｐｏｓｉｔｉｖｅ　ｓｅｌｅｃｔｉｏｎ　ｋｉｔ（Ｓｔｅｍｃｅｌｌ　Ｔｅｃｈｎｏｌｏｇｉｅｓ　Ｉｎｃ，Ｖａｎｃｏｕｖｅｒ，Ｃａｎａｄａ，Ｃａｔ．Ｎｏ．２００００）を用いて、ＲＯＢＯＳＥＰ（Ｓｔｅｍｃｅｌｌ　Ｔｅｃｈｎｏｌｏｇｉｅｓ　Ｉｎｃ，Ｖａｎｃｏｕｖｅｒ，Ｃａｎａｄａ，Ｃａｔ．Ｎｏ．２０１０４）により、ＣＤ１９陽性の細胞を分離した。遠心分離後（１２００ｒｐｍ、４℃、５分）、５×１０５細胞／ｍｌになるようにＲＰＭＩ１６４０培地（１０％　ＦＣＳ、５０　Ｕ／ｍｌ　Ｐ－Ｓ、１×１０－４　Ｍ　２－ＭＥ含有）に懸濁した。５００Ｕ／ｍｌ　ｒｍＩＬ－４（Ｒ＆Ｄ　ｓｙｓｔｅｍ，　Ｉｎｃ．，　Ｍｉｎｎｅａｐｏｌｉｓ，　ＵＳＡ，　Ｃａｔ．Ｎｏ．４０４－ＭＬ－０１０）と１０μｇ／ｍｌ　ＬＰＳ（Ｓｉｇｍａ，　Ｓｔ．Ｌｏｕｉｓ，　ＵＳＡ，　Ｃａｔ．Ｎｏ．Ｌ６１４３）とともに、各濃度の試料を添加し、９６ウェルプレートに播種した（２００μｌ／ｗｅｌｌ）。対照群として、試料を添加しない群を設けた。３７℃、５％　ＣＯ２条件下で９日間培養した後、培養液上清を回収し、ＥＬＩＳＡにより、ＩｇＥ濃度を測定した。 Test Method Spleens were isolated from NC / Tnd mice (7-9 weeks old) in an SPF environment, homogenized in RPMI 1640 medium, and a single cell suspension was prepared with a 70 μm mesh filter. After centrifugation (1200 rpm, 4 ° C., 5 minutes), the suspension was resuspended in Robosp buffer (Stemcell Technologies Inc, Vancouver, Canada, Cat. No. 20104) to a concentration of 1 × 10 8 cells / ml. Using Mouse CD19 positive selection kit (Stemcell Technologies Inc, Vancouver, Canada, Cat. No. 20000), ROBOSEP (Stemcell Technologies Inc., Candoc. After centrifugation (1200 rpm, 4 ° C., 5 minutes), RPMI1640 medium (containing 10% FCS, 50 U / ml PS, 1 × 10 −4 M 2-ME) to 5 × 10 5 cells / ml It was suspended in. 500 U / ml rmIL-4 (R & D system, Inc., Minneapolis, USA, Cat. No. 404-ML-010) and 10 μg / ml LPS (Sigma, St. Louis, USA, Cat. No. L6143) Concentration samples were added and seeded in 96-well plates (200 μl / well). As a control group, a group to which no sample was added was provided. After culturing at 37 ° C. under 5% CO 2 for 9 days, the culture supernatant was collected and the IgE concentration was measured by ELISA.
・結果
　マウス脾臓より単離したＢ細胞に対して、マンゴスチン果皮抽出物、α－マンゴスチン、γ－マンゴスチンはＩＬ－４とＬＰＳにより誘導されるＩｇＥの産生を抑制した。結果を表１及び図２に示す。ＩＣ５０はそれぞれ４．８μｇ／ｍｌ、２．５μＭ、１．６μＭであった。 -Results On B cells isolated from mouse spleen, mangosteen peel extract, α-mangosteen and γ-mangostin suppressed the production of IgE induced by IL-4 and LPS. The results are shown in Table 1 and FIG. The IC50 was 4.8 μg / ml, 2.5 μM, and 1.6 μM, respectively.
　Ｉｎ　ｖｉｔｒｏにおけるサイトカイン産生に対する影響（メッセンジャーＲＮＡ（ｍＲＮＡ）レベルでの解析）
・試験法
　コンベンショナル環境下で皮膚炎を発症したＮＣ／Ｔｎｄマウス（１２～１７週齢）から、腋窩リンパ節を単離し、ＲＰＭＩ１６４０培地中でホモジェナイズ後、７０μｍメッシュのフィルターにより、単一細胞懸濁液を作製した。遠心分離後（１２００ｒｐｍ、４℃、５　ｍｉｎ）、２×１０６細胞／ｍｌの濃度になるように、ＲＰＭＩ１６４０培地（１０％　ＦＣＳ、５０Ｕ／ｍｌ　Ｐ－Ｓ、１×１０－４Ｍ　２－ＭＥ含有）に懸濁した。１μｇ／ｍｌポークウィードマイトジェン（ＰＷＭ；Ｓｉｇｍａ，Ｓｔ．Ｌｏｕｉｓ，ＵＳＡ，Ｃａｔ．Ｎｏ．Ｌ６１４３）とともに、各濃度のサンプルを添加し、２４ウェルプレートに播種し（１ｍｌ／ｗｅｌｌ）、３７℃、５％ＣＯ２条件下で２４時間培養した。対照群として、ＰＷＭを添加しない群(図３、４では培地と示す)、また、ＰＷＭのみを添加してサンプルを添加しない群を設けた（図３、４ではPWMと示す）。その後、ＴＡＫＡＲＡ　ＦａｓｔＰｕｒｅ　ＲＮＡ　ｋｉｔ（ＴＡＫＡＲＡ　Ｂｉｏ　Ｉｎｃ．，　Ｓｈｉｇａ，Ｊａｐａｎ，Ｃａｔ．Ｎｏ．９１９０）により、トータルＲＮＡを単離し、ＴＡＫＡＲＡ　ＰｒｉｍｅＳｃｒｉｐｔ　１ｓｔ　ｓｔｒａｎｄ　ｃＤＮＡ　Ｓｙｎｔｈｅｓｉｓ　ｋｉｔ（ＴＡＫＡＲＡ　Ｂｉｏ　Ｉｎｃ．，Ｓｈｉｇａ，Ｊａｐａｎ，Ｃａｔ．Ｎｏ．６１１０Ａ）を用いてｃＤＮＡの作製を行い、リアルタイムＰＣＲを行った。なお、内在性コントロールとしてβ－ａｃｔｉｎを使用し、比較ＣＴ法（ΔΔＣＴ法）による比較を行った。ΔΔＣＴ法は、１サイクルの検出の違いで、発現量に２倍量の差があるという理論をもとに、基準となる群と比較して、調べたい群が何サイクル早く、もしくは何サイクル遅くＴｈｒｅｓｈｏｌｄ　Ｌｉｎｅに達するかに注目して、発現量を相対定量する方法である。ΔΔＣＴ法においては、ＰＷＭを添加しない群（培地）を基準にした。 Effect on cytokine production in vitro (analysis at messenger RNA (mRNA) level)
Test method: Axillary lymph nodes were isolated from NC / Tnd mice (12-17 weeks old) that developed dermatitis in a conventional environment, homogenized in RPMI 1640 medium, and then suspended in a single cell with a 70 μm mesh filter. A liquid was prepared. After centrifugation (1200 rpm, 4 ° C., 5 min), RPMI1640 medium (10% FCS, 50 U / ml PS, 1 × 10 −4 M 2-ME) to a concentration of 2 × 10 6 cells / ml Contained). Samples of each concentration were added together with 1 μg / ml pork weed mitogen (PWM; Sigma, St. Louis, USA, Cat. No. L6143), seeded in a 24-well plate (1 ml / well), 37 ° C., 5% Culturing for 24 hours under CO 2 conditions. As a control group, a group to which no PWM was added (shown as a medium in FIGS. 3 and 4) and a group to which only PWM was added and no sample was added (shown as PWM in FIGS. 3 and 4) were provided. Thereafter, total RNA was isolated by TAKARA FastPure RNA kit (TAKARA Bio Inc., Shiga, Japan, Cat. No. 9190), and TAKARA PrimeScript 1st strand cDNA Synthesis kit (TAKARAJ. 6110A), cDNA was prepared and real-time PCR was performed. Note that β-actin was used as an endogenous control, and a comparison was made by a comparative CT method (ΔΔCT method). The ΔΔCT method is based on the theory that there is a double amount difference in the expression level due to the difference in detection in one cycle. It is a method for relative quantification of the expression level, paying attention to whether it reaches the Threshold Line. In the ΔΔCT method, a group (medium) not added with PWM was used as a reference.
・結果
　結果を図３，４に示す。マウス腋下リンパ節より単離したリンパ球に対して、マンゴスチン果皮抽出物、α－マンゴスチン、γ－マンゴスチンはＰＷＭにより誘導されるＩＦＮ－γのｍＲＮＡの発現上昇を抑制した。ＩＣ５０はそれぞれ６．２μｇ／ｍｌ、３．６μＭ、３．６μＭであった。一方、ＩＬ－４，ＩＬ－５，ＩＬ－１３に対しては有意な抑制を示さなかった。 -Results The results are shown in FIGS. Mangosteen peel extract, α-mangostin, and γ-mangostin suppressed the increase in IFN-γ mRNA expression induced by PWM on lymphocytes isolated from mouse mammary lymph nodes. The IC50 was 6.2 μg / ml, 3.6 μM and 3.6 μM, respectively. On the other hand, no significant inhibition was shown for IL-4, IL-5, and IL-13.
　Ｉｎ　ｖｉｔｒｏにおけるＴＳＬＰ産生に対する影響（ｍＲＮＡレベルでの解析）
・試験法
　ＮＣ／Ｔｎｄマウス新生児（１－３日）を屠殺し、皮膚をハサミで切り取った。皮下脂肪を物理的に除去した後、６ｍｍパンチにより皮膚サンプルを得た。０．２５％トリプシン－ＥＤＴＡ溶液に皮膚サンプルを浮かせ、４℃で１時間培養した後、３７℃で３０分培養した。ピンセットを用いて皮膚サンプルより表皮を単離し、２４ウェルプレートにおいて培地に浮かせた。様々な濃度の試薬を添加し、３７℃で１時間、前培養した後、ＴＮＦ－α（２５ｎｇ／ｍｌ）、ＩＬ－１β（１０ｎｇ／ｍｌ）を添加し３７℃、５％ＣＯ２条件下で培養した。対照として、試薬もＴＮＦ―α、ＩＬ－１βも加えない群（図５中最も左側）、ＴＮＦ―α、ＩＬ－１βのみを加えた群（図５中左から２番目）を設けた。２４時間後、Ｔａｋａｒａ　ＲＮＡ　Ｆａｓｔｐｕｒｅを用いてトータルＲＮＡを抽出し、リアルタイムＰＣＲを行った。なお、内在性コントロールとしてβ－ａｃｔｉｎを使用し、ΔΔＣＴ法による比較を行った。ΔΔＣＴ法においては、試薬もＴＮＦ―α、ＩＬ－１βも加えない群を基準にした。 Effect on TSLP production in vitro (analysis at the mRNA level)
Test Method NC / Tnd mouse neonates (1-3 days) were sacrificed and the skin was cut with scissors. After physically removing the subcutaneous fat, a skin sample was obtained with a 6 mm punch. The skin sample was floated on a 0.25% trypsin-EDTA solution, incubated at 4 ° C. for 1 hour, and then incubated at 37 ° C. for 30 minutes. The epidermis was isolated from the skin sample using tweezers and floated on the medium in a 24-well plate. After adding various concentrations of reagents and pre-incubating at 37 ° C. for 1 hour, TNF-α (25 ng / ml) and IL-1β (10 ng / ml) were added, and at 37 ° C. under 5% CO 2 conditions. Cultured. As a control, a group to which neither a reagent nor TNF-α and IL-1β was added (the leftmost in FIG. 5) and a group to which only TNF-α and IL-1β were added (the second from the left in FIG. 5) were provided. After 24 hours, total RNA was extracted using Takara RNA Fastpure, and real-time PCR was performed. Note that β-actin was used as an endogenous control, and comparison was made by the ΔΔCT method. In the ΔΔCT method, a group to which no reagent, TNF-α, and IL-1β were added was used as a reference.
・結果
　結果を図５に示す。表皮組織培養系において、ＴＮＦ－αとＩＬ－１βの共添加により誘導されるＴＳＬＰのｍＲＮＡの発現上昇に対して、マンゴスチン果皮抽出物、α－マンゴスチンは発現抑制作用を示した。 -Results The results are shown in FIG. In the epidermal tissue culture system, mangosteen peel extract and α-mangostin showed an expression-inhibiting action against the increase in TSLP mRNA expression induced by co-addition of TNF-α and IL-1β.
・試験法
　コンベンショナル環境下で未だ皮膚炎を発症していないＮＣ／Ｔｎｄマウス（５－６週齢）を、対照飼料投与群、マンゴスチン果皮抽出物を含む混餌飼料の投与群であるマンゴスチン投与群との２群（各群ｎ＝７）に分けた。いずれの群も、コンベンショナル環境下で対照飼料を用いて１週間の予備飼育を行った後、それぞれの試験飼料を自由摂取させた。すなわち、マンゴスチン投与群には、マンゴスチン果皮抽出物を０．２５％含む混餌飼料を与え（マンゴスチン果皮抽出物の摂取量は、２５０ｍｇ／ｋｇ／ｄａｙとなる）、対照試料投与群には、ＣＲＦ－１（オリエンタル酵母社）を投与した。試験飼料を６週間投与した後、頸椎脱臼によりマウスを屠殺し、６ｍｍパンチにより、皮膚サンプルを得た。
　得られた皮膚サンプルは、ＴＡＫＡＲＡ　ＦａｓｔＰｕｒｅ　ＲＮＡ　ｋｉｔ（ＴＡＫＡＲＡ　Ｂｉｏ　Ｉｎｃ．，　Ｓｈｉｇａ，Ｊａｐａｎ，Ｃａｔ．Ｎｏ．９１９０）を用いてトータルＲＮＡを単離し、ＴＡＫＡＲＡ　ＰｒｉｍｅＳｃｒｉｐｔ　１ｓｔ　ｓｔｒａｎｄ　ｃＤＮＡ　Ｓｙｎｔｈｅｓｉｓ　ｋｉｔ（ＴＡＫＡＲＡ　Ｂｉｏ　Ｉｎｃ．，Ｓｈｉｇａ，Ｊａｐａｎ，Ｃａｔ．Ｎｏ．６１１０Ａ）を用いてｃＤＮＡの作製を行い、様々なサイトカインやケモカイン、神経線維の伸展調節に関わるＮＧＦやセマフォリン３ａ（Ｓｅｍａ３ａ）、細胞増殖制御因子であるＡＱＰ３のｍＲＮＡ発現量を解析するためリアルタイムＰＣＲを行った。なお、内在性コントロールとしてβ－ａｃｔｉｎを使用し、ΔΔＣＴ法による比較を行った。ΔΔＣＴ法においては、対照飼料群を基準にした。 Test method NC / Tnd mice (5-6 weeks old) who have not yet developed dermatitis in a conventional environment, a control diet administration group, a mangosteen administration group which is an administration group of a mixed diet containing mangosteen peel extract, Into two groups (each group n = 7). All groups were allowed to freely ingest each test feed after a one-week preliminary breeding using a control feed in a conventional environment. That is, the mangosteen administration group was given a mixed diet containing 0.25% of mangosteen peel extract (the intake of mangosteen peel extract was 250 mg / kg / day), and the control sample administration group had CRF- 1 (Oriental Yeast) was administered. After administration of the test feed for 6 weeks, the mice were sacrificed by cervical dislocation and skin samples were obtained by 6 mm punch.
From the obtained skin sample, total RNA was isolated using TAKARA FastPure RNA kit (TAKARA Bio Inc., Shiga, Japan, Cat. No. 9190), and TAKARA PrimeScript 1st strand cDNA Synthesis Science. , Japan, Cat.No. 6110A), and mRNA expression of various cytokines, chemokines, NGF and semaphorin 3a (Sema3a) involved in the regulation of nerve fiber extension, and AQP3, a cell growth regulator. Real-time PCR was performed to analyze the quantity. Note that β-actin was used as an endogenous control, and comparison was made by the ΔΔCT method. In the ΔΔCT method, the control feed group was used as a reference.
・結果
　結果を図６に示す。図６において、縦軸は対照飼料群での値を１とした相対値である。マンゴスチン果皮抽出物投与群では、皮膚のＴＳＬＰ、ＩＬ－４、ＩＦＮ－γ、ＣＣＬ２２（ＭＤＣ）、ＣＣＬ２４（Ｅｏｔａｘｉｎ－２）、ＮＧＦ、ＡＱＰ３のｍＲＮＡの発現量が有意に低かった。 -Results The results are shown in FIG. In FIG. 6, the vertical axis is a relative value where the value in the control feed group is 1. In the mangosteen peel extract administration group, the expression levels of TSLP, IL-4, IFN-γ, CCL22 (MDC), CCL24 (Eotaxin-2), NGF, and AQP3 mRNA in the skin were significantly low.
　この出願は２０１２年７月１９日に出願された日本国特許出願第２０１２－１６０４２９号からの優先権を主張するものであり、その内容を引用してこの出願の一部とするものである。

  This application claims priority from Japanese Patent Application No. 2012-160429 filed on July 19, 2012, the contents of which are incorporated herein by reference.

Claims (13)

  1. 　マンゴスチン果皮抽出物からなる免疫調節剤。 An immunomodulator consisting of mangosteen peel extract.
  2. 　細胞の増殖を抑制することを特徴とする請求項１に記載の免疫調節剤。 The immunomodulator according to claim 1, which suppresses cell proliferation.
  3. 　ＩＦＮ－γの産生を抑制することを特徴とする請求項１から２のいずれかに記載の免疫調節剤。 The immunomodulator according to any one of claims 1 to 2, which suppresses the production of IFN-γ.
  4. 　ＴＳＬＰの産生を抑制することを特徴とする請求項１から３のいずれかに記載の免疫調節剤。 The immunomodulator according to any one of claims 1 to 3, which suppresses the production of TSLP.
  5. 　細胞が、ケラチノサイト、肥満細胞及びＢ細胞からなる群から選ばれる１種類の細胞であることを特徴とする請求項２あるいは請求項２に係る請求項３または４のいずれかに記載の免疫調節剤。 The immunomodulator according to any one of claims 2 and 2, wherein the cell is one type of cell selected from the group consisting of keratinocytes, mast cells, and B cells. .
  6. 　ＩＬ－４、ＣＣＬ２２（ＭＤＣ）、ＣＣＬ２４（Ｅｏｔａｘｉｎ－２）、ＮＧＦ、ＡＱＰ３のいずれか少なくとも一種類の産生を抑制することを特徴とする請求項１から５のいずれか１項に記載の免疫調節剤。 The immunoregulation according to any one of claims 1 to 5, which suppresses production of at least one of IL-4, CCL22 (MDC), CCL24 (Eotaxin-2), NGF, and AQP3. Agent.
  7. 　α－マンゴスチンからなる免疫調節剤。 An immunomodulator consisting of α-mangosteen.
  8. 　細胞の増殖を抑制することを特徴とする請求項７に記載の免疫調節剤。 The immunomodulator according to claim 7, which suppresses cell proliferation.
  9. 　ＩｇＥ、ＩＦＮ－γまたはＴＳＬＰのいずれか少なくとも一種類の産生を抑制することを特徴とする請求項７または８のいずれかに記載の免疫調節剤。 The immunomodulator according to claim 7 or 8, which suppresses production of at least one of IgE, IFN-γ and TSLP.
  10. 　γ－マンゴスチンからなる免疫調節剤。 An immunoregulator comprising γ-mangosteen.
  11. 　細胞の増殖を抑制することを特徴とする請求項１０に記載の免疫調節剤。 The immunomodulator according to claim 10, which suppresses cell proliferation.
  12. 　ＩｇＥまたはＩＦＮ－γのいずれか少なくとも一種類の産生を抑制することを特徴とする請求項１０または１１のいずれかに記載の免疫調節剤。 The immunomodulator according to claim 10 or 11, which suppresses production of at least one of IgE and IFN-γ.
  13. 　請求項１から１２のいずれかに記載の免疫調節剤を含む、免疫調節のための飲食品。
     

          Food-drinks for immunomodulation containing the immunomodulator in any one of Claims 1-12.


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