JPWO2008105540A1 - Drugs and foods effective for allergic diseases and autoimmune diseases - Google Patents

Abstract

The present invention provides pharmaceuticals and foods capable of effectively and safely treating, improving or preventing allergic diseases such as type I allergic diseases and autoimmune diseases such as rheumatoid arthritis and inflammatory bowel disease. For the purpose. That is, the present invention relates to a pharmaceutical comprising a fermented grape skin / seed lactic acid bacteria obtained by fermenting at least one of grape skin and seeds with lactic acid bacteria, and at least one of grape skins and seeds It is related with the foodstuff characterized by including the grape skin and seed lactic-acid-bacteria fermented material obtained by fermenting lactic acid bacteria with lactic acid bacteria.

Description

  The present invention relates to a medicine and a food using at least one of grape skin and seed for the purpose of treating, improving or preventing allergic diseases and autoimmune diseases.

  The population suffering from allergic diseases is considered to be about one third of the national population, and is increasing even though many therapeutic drugs and treatments have been introduced. Allergies are classified into types I to IV, and hay fever, asthma, atopic dermatitis and the like belong to type I allergies. Type I allergy develops by the following mechanism. That is, Th2 cells induced by antigen sensitization differentiate B cells into IgE antibody-producing cells, and the produced IgE antibodies bind to mast cells. When the antigen binds to the IgE antibody on the mast cell, degranulation occurs and an inflammatory substance such as histamine is released, and an immediate allergic reaction such as redness, swelling, and airway contraction is induced. Furthermore, after degranulation of mast cells, Th2 cells are activated by cytokines released from mast cells, and eosinophils are mobilized and activated by the cytokines produced from Th2 cells. Activated eosinophils release inflammatory substances, resulting in a more severe late-onset inflammation.

  Rheumatoid arthritis is an extremely important disease affecting approximately 1% of the world population, and its etiology and pathogenesis mechanism have not been completely clarified yet. However, rheumatoid arthritis is self-induced due to Th1 cells induced by self-antigens. It is considered an immune disease. Tumor necrosis factor (TNF-α), interleukin 1 (IL-1), IL-6, prostaglandin E2 (PGE2), nitric oxide (NO), etc. produced from macrophages activated by Th1 cells Inflammatory substances cause chronic inflammation in the joint synovium and joint destruction occurs. In addition to these antigen-specific inflammatory reactions, there are also antigen-nonspecific inflammatory reactions that are induced by inflammatory agents such as carrageenan, which are activated when macrophages are directly stimulated by inflammatory agents, Induced by producing inflammatory substances. That is, in such reactions, macrophages work as effector cells as in autoimmune diseases such as rheumatoid arthritis.

  Inflammatory bowel diseases such as ulcerative colitis and Crohn's disease cause inflammation in the intestinal tract, exhibiting symptoms such as severe diarrhea and bloody stool, and symptoms such as weight loss and anemia. In addition, it is known that the incidence of colorectal cancer is increased in ulcerative colitis. Although the cause of the onset is unknown, it is considered to be an autoimmune disease caused by the failure of immune tolerance against resident bacteria in the autointestinal tract, and inflammation such as TNF-α produced from activated macrophages Sex cytokines are the cause of injury (see, for example, Non-Patent Document 1). Salazopyrine, an anti-inflammatory drug, has been used as a drug treatment, but it has not been completely cured, and steroids and immunosuppressants are used as symptoms worsen. Side effects are a problem. Ulcerative colitis is designated as a specific disease by the Ministry of Health, Labor and Welfare, and according to the number of specific diseases received in 2006, the number of patients with ulcerative colitis is the largest, about 90,000, and it tends to increase every year. Is seen.

  Lactic acid bacteria have been shown to be effective against type I allergic reactions such as hay fever and atopic dermatitis, and the mechanism of action is attributed to suppression of Th2 cells by activation of Th1 cells (for example, Non-patent document 2). It is considered that administration of lactic acid bacteria from the antigen sensitization stage suppresses the induction of Th2 cells, suppresses subsequent IgE antibody production, and consequently suppresses allergic reactions. However, after mast cells are sensitized by the produced IgE antibody, the degranulation reaction caused by the antigen binding to the mast cells has no inhibitory effect on lactic acid bacteria, and its effectiveness has not been shown. That is, lactic acid bacteria are effective when administered prophylactically before the onset of allergy, but are not effective when administered for therapeutic purposes after onset. In addition, lactic acid bacteria are not effective for autoimmune diseases induced by Th1 cells.

  In recent years, it has been reported that flavonoids contained in a large amount in the skin and seeds of plants have various physiological activities. In general, flavonoids are thought to act on effector cells and exert anti-inflammatory effects. Examples of the action of flavonoids on such an inflammatory reaction include degranulation inhibitory action using a mast cell line in an in vitro test (for example, see Non-Patent Document 3), and suppression of inflammatory cytokine production using a macrophage cell line. Effects (for example, see Non-Patent Document 4) have been reported. In in vivo tests, it has been reported that intraperitoneal administration of flavonoids suppresses passive skin anaphylaxis (PCA) reaction, which is a degranulation reaction of mast cells (see, for example, Non-Patent Document 5). However, many reports on the effects of conventional flavonoids are based on in vitro tests, and even in in vivo tests, there are evaluations by tests such as intraperitoneal administration and intravenous administration, but there are very few evaluations by oral administration. . Moreover, it has not been reported that flavonoids act during antigen sensitization like lactic acid bacteria and suppress antigen-specific IgE antibody production.

  As described above, various methods have been proposed for treating, ameliorating, or preventing allergic diseases and autoimmune diseases, but the number of patients suffering from these diseases is still large and more effective. At present, there is still a need for safe treatment, improvement, or prevention methods.

Reinacker H. C. et. al. , Clin. Exp. Immunol. 94,174-181.1993 J. et al. Dairy Sci. 1998; 81: 48-53. Biosci. Biotechnol. Biocem. 1998; 62: 1284-1289. Biosci. Biotechnol. Biocem. 2004; 67: 1549-1557. Planta. Med. 2005; 71: 24-27.

  An object of the present invention is to solve the conventional problems and achieve the following objects. That is, an object of the present invention is to provide a medicine and a food that can effectively, safely treat, ameliorate, or prevent allergic diseases and autoimmune diseases.

In order to solve the above-mentioned problems, the present inventors have made extensive studies and as a result, obtained the following findings. That is, a fermented grape skin / seed lactic acid bacterium obtained by fermenting at least one of grape skin and seed with lactic acid bacteria has an excellent inhibitory effect on vascular permeability, IgE antibody production, eosinophil infiltration, anti-inflammatory Therefore, allergic diseases and autoimmune diseases can be effectively and safely treated, improved, or prevented by using the grape skin / seed lactic acid bacteria fermented product. It is a knowledge that can be done.
The inventors of the present invention have provided that a fermented grape skin / seed lactic acid bacteria obtained by fermenting grape skin and seeds with lactic acid bacteria suppresses antigen-specific IgE antibody production induced by ovalbumin antigen in an allergy model of mice, It was found that eosinophil infiltration into the bronchus and alveoli after induction was suppressed. In addition, we found that fermented grape skin and seed lactic acid bacteria suppress passive skin anaphylaxis (PCA) reaction in mice passively sensitized with IgE antibody. Furthermore, the present inventors have found that a fermented grape skin / seed lactic acid bacterium suppresses foot edema induced by carrageenan as an inflammation agent in a mouse rheumatoid arthritis model, and induces arthritis induced by sensitization with type II collagen. It was found to suppress the onset of. Furthermore, the present inventors have found that a fermented grape skin / seed lactic acid bacterium suppresses an increase in TNF-α production and alleviates symptoms of ulcerative colitis in a mouse ulcerative colitis model ( See Examples, below).
From these results, the present inventors have found that allergic diseases and autoimmune diseases can be effectively and safely treated, ameliorated, or prevented by using a grape peel / seed lactic acid bacteria fermented product. It was completed.

The present invention is based on the above findings by the present inventors, and means for solving the above problems are as follows. That is,
<1> A medicament for treating or preventing at least one of allergic diseases and autoimmune diseases, wherein a fermented grape skin / seed lactic acid bacteria is obtained by fermenting at least one of grape skin and seeds with lactic acid bacteria. It is a medicine characterized by containing.
<2> The medicament according to <1>, including a fermented grape skin / seed lactic acid bacteria obtained by fermenting at least one of grape skin and seeds in a dried state with lactic acid bacteria.
<3> In any one of the above items <1> to <2>, including a fermented grape skin / seed lactic acid bacterium obtained by fermenting at least one of grape skin and seed in a fine powder (powder) state with lactic acid bacteria It is the medicine described.
<4> Any one of <1> to <3>, including a grape skin / seed lactic acid bacteria fermented product obtained by fermenting at least one of grape skin and seed in a dry powder (dried powder) state with lactic acid bacteria It is the medicine described.
As <5> lactic acid bacteria, Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus salivarius (Lactobacillus salivalius), Lactobacillus amylovorus (Lactobacillus amylovorus), Lactobacillus casei (Lactobacillus casei), Rakutobachiru Kitasatonisu (Lactobacillus kitasatonisi), Rakutobachiru reuteri (Lactobacillus reuteri), Lactobacillus jonsonii, Lactobacillus acidophilus, Lactobacillus brevis (Lactobac) llus brevis), Lactobacillus Korinihorumisu (Lactobacillus coryniformis), Lactobacillus Karubatasu (Lactobacillus curvatus), Lactobacillus Deruburyukyi (Lactobacillus delbrueckii), Lactobacillus Farsi Minas (Lactobacillus farciminus), Lactobacillus rhamnosus (Lactobacillus rhamnosus), Pediococcus Damunosasu ( Pediococcus damnosus, Pediococcus halophyllus, Pediococcus parvulus, Pediococcus pe Tosaseus (Pediococcus pentocaseus), Bifidobacterium adrecentis (Bifidobacterium adolecentis), Bifidobacterium bifidus ((Bifidobacterium bifidus), Lactococcus cremolith (Lactococcus cremolicus) oenos), Leuconostoc lactis, Leuconostoc mesenteroides, and Enterococcus faecis Wherein comprising at least grape skin, seed lactobacillus fermentation product obtained by using one selected from the group consisting of lis) <1> is a medicament according to any one of <4>.
<6> The fermented product of grape skin / seed lactic acid bacteria is an organic solvent extract obtained by further extracting the fermented product of grape skins / seed lactic acid bacteria with an organic solvent, according to any one of <1> to <5>. It is a medicine.
<7> The pharmaceutical according to any one of <1> to <6>, wherein the fermented grape skin / seed lactic acid bacteria is an acetone extract obtained by further extracting the fermented grape skin / seed lactic acid bacteria with acetone. It is.
<8> The above <1> to <7>, wherein the grape skin / seed lactic acid bacteria fermented product is a hydrophobic substance contained in an acetone extract obtained by further extracting the grape skin / seed lactic acid bacteria fermented product with acetone. Or a pharmaceutical according to any one of the above.
<9> Grape peel / seed lactic acid bacteria fermentation product is a hydrophobic substance contained in an acetone extract obtained by further extracting the grape skin / seed lactic acid bacteria fermentation product with acetone, and gel filtration of the hydrophobic substance The pharmaceutical according to any one of <1> to <8>, wherein the molecular weight by chromatography is in the range of 100 to 1,000.
<10> Grape peel / seed lactic acid bacteria fermentation product is a hydrophobic substance contained in an acetone extract obtained by further extracting the grape skin / seed lactic acid bacteria fermentation product with acetone, and gel filtration of the hydrophobic substance Chromatographic molecular weight is in the range of 100 to 1,000, silica gel is used as an adsorbent, and chloroform-methanol mixed solvent having a volume ratio of chloroform to methanol of 15: 1 is used as a developing solvent. The pharmaceutical composition according to any one of <1> to <9>, wherein the Rf value in the thin-layer chromatography is in the range of 0.3 to 0.4.
<11> Grape peel / seed lactic acid bacteria fermentation product is a hydrophobic substance contained in an acetone extract obtained by further extracting the grape skin / seed lactic acid bacteria fermentation product with acetone, and gel filtration of the hydrophobic substance Chloroform-methanol whose molecular weight by chromatography is in the range of 100 to 1,000, silica gel is used as the adsorbent, and volume ratio of chloroform, methanol and ethyl acetate is 15: 1: 1 as the developing solvent -The pharmaceutical according to any one of <1> to <9>, wherein the Rf value in thin layer chromatography when using an ethyl acetate mixed solvent is in the range of 0.4 to 0.5.
<12> The medicament according to any one of <1> to <11>, which is used for treatment or prevention of type I allergic disease.
<13> The medicament according to any one of <1> to <11>, which is used for treatment or prevention of rheumatoid arthritis.
<14> The medicament according to any one of <1> to <11>, which is used for treatment or prevention of inflammatory bowel disease.
<15> The medicament according to any one of <1> to <14>, which has a blood vessel permeability enhancement inhibitory action.
<16> The medicament according to any one of <1> to <14>, which has an IgE antibody production inhibitory action.
<17> The medicament according to any one of <1> to <14>, which has an inhibitory action on eosinophil infiltration.
<18> The medicament according to any one of <1> to <14>, which has an anti-inflammatory action (for example, an edema inhibitory action, a swelling inhibitory action).
<19> The medicament according to any one of <1> to <14>, which has an inhibitory action on TNF-α production.

<20> A food for improving or preventing at least one of allergic diseases and autoimmune diseases, wherein a fermented grape skin / seed lactic acid bacteria obtained by fermenting at least one of grape skin and seeds with lactic acid bacteria It is a food characterized by containing.
<21> The food according to <20>, including a fermented grape skin / seed lactic acid bacteria obtained by fermenting at least one of grape skin and seeds in a dried state with lactic acid bacteria.
<22> Any one of the above <20> to <21>, including a grape skin / fermented lactic acid bacteria fermented product obtained by fermenting at least one of grape skin and seeds in a fine powder (powder) state with lactic acid bacteria It is the described food.
<23> The above <20> to <22>, which includes a grape skin / fermented lactic acid bacteria fermented product obtained by fermenting at least one of grape skin and seeds in a dry powder (dried powder) state with lactic acid bacteria It is the described food.
As <24> lactic acid bacteria, Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus salivarius (Lactobacillus salivalius), Lactobacillus amylovorus (Lactobacillus amylovorus), Lactobacillus casei (Lactobacillus casei), Rakutobachiru Kitasatonisu (Lactobacillus kitasatonisi), Rakutobachiru reuteri (Lactobacillus reuteri), Lactobacillus jonsonii, Lactobacillus acidophilus, Lactobacillus brevis (Lactoba) illus brevis), Lactobacillus Korinihorumisu (Lactobacillus coryniformis), Lactobacillus Karubatasu (Lactobacillus curvatus), Lactobacillus Deruburyukyi (Lactobacillus delbrueckii), Lactobacillus Farsi Minas (Lactobacillus farciminus), Lactobacillus rhamnosus (Lactobacillus rhamnosus), Pediococcus Damunosasu ( Pediococcus damnosus), Pediococcus halophyllus, Pediococcus parvulus, Pediococcus Pentiococcus pentoaceus, Bifidobacterium adrecentis (Bifidobacterium adolecentis), Bifidobacterium bifidus ((Bifidobacterium coficus) Lactococcus cremolith (Lactococcus cremolicus) oenos), Leuconostoc lactis, Leuconostoc mesenteroides, and Enterococcus faecalis Wherein comprising at least grape skin, seed lactobacillus fermentation product obtained by using one selected from the group consisting of alis) from <20> is a food product according to any one of <23>.
<25> The fermented grape skin / seed lactic acid bacteria is an organic solvent extract obtained by further extracting the fermented grape skin / seed lactic acid bacteria with an organic solvent, according to any one of <20> to <24>. Food.
<26> The food according to any one of <20> to <25>, wherein the fermented grape skin / seed lactic acid bacteria is an acetone extract obtained by further extracting the fermented grape skin / seed lactic acid bacteria with acetone. It is.
<27> Grape peel / seed lactic acid bacteria fermentation product is a hydrophobic substance contained in an acetone extract obtained by further extracting the grape skin / seed lactic acid bacteria fermentation product with acetone. <20> to <26> It is a foodstuff in any one of.
<28> Grape peel / seed lactic acid bacteria fermentation product is a hydrophobic substance contained in an acetone extract obtained by further extracting the grape skin / seed lactic acid bacteria fermentation product with acetone, and gel filtration of the hydrophobic substance The food according to any one of <20> to <27>, wherein the molecular weight by chromatography is in the range of 100 to 1,000.
<29> Grape peel / seed lactic acid bacteria fermentation product is a hydrophobic substance contained in an acetone extract obtained by further extracting the grape skin / seed lactic acid bacteria fermentation product with acetone, and gel filtration of the hydrophobic substance Chromatographic molecular weight is in the range of 100 to 1,000, silica gel is used as an adsorbent, and chloroform-methanol mixed solvent having a volume ratio of chloroform to methanol of 15: 1 is used as a developing solvent. In the above-mentioned <20> to <28>, the Rf value in thin-layer chromatography is in the range of 0.3 to 0.4.
<30> Grape peel / seed lactic acid bacteria fermentation product is a hydrophobic substance contained in an acetone extract obtained by further extracting the grape skin / seed lactic acid bacteria fermentation product with acetone, and gel filtration of the hydrophobic substance Chloroform-methanol whose molecular weight by chromatography is in the range of 100 to 1,000, silica gel is used as the adsorbent, and volume ratio of chloroform, methanol and ethyl acetate is 15: 1: 1 as the developing solvent -The food according to any one of <20> to <28>, wherein the Rf value in thin layer chromatography when using an ethyl acetate mixed solvent is in the range of 0.4 to 0.5.
<31> The food according to any one of <20> to <30>, which is used for improving or preventing a type I allergic disease.
<32> The food according to any one of <20> to <30>, which is used for improving or preventing rheumatoid arthritis.
<33> The food according to any one of <20> to <30>, which is used for improvement or prevention of inflammatory bowel disease.
<34> The food according to any one of <20> to <33>, which has a blood vessel permeability enhancement inhibitory effect.
<35> The food according to any one of <20> to <33>, which has an IgE antibody production inhibitory effect.
<36> The food according to any one of <20> to <33>, which has an action of suppressing eosinophil infiltration.
<37> The food according to any one of <20> to <33>, which has an anti-inflammatory action (for example, an edema inhibitory action and a swelling inhibitory action).
<38> The food according to any one of <20> to <33>, which has a TNF-α production inhibitory action.

  According to the present invention, it is possible to solve the conventional problems, achieve the object, and to effectively and safely treat, improve, or prevent allergic diseases and autoimmune diseases and Food can be provided.

1A is a graph showing the passive skin anaphylaxis (PCA) inhibitory effect of a grape skin / seed lactic acid bacteria fermented product sample in Example 1. FIG. FIG. 1B is a graph showing the passive skin anaphylaxis (PCA) inhibitory action of specimens other than the grape skin / seed lactic acid bacteria fermented product in Example 1. FIG. 2 is a graph showing the IgE production inhibitory effect of the grape skin / seed lactic acid bacteria fermented material sample in Example 2. FIG. 3 is a graph showing the action of inhibiting eosinophil infiltration into the bronchi and alveoli of a grape skin / seed lactic acid bacteria fermented product sample in Example 3. FIG. 4 is a graph showing the carrageenan-induced mouse foot edema inhibitory action of grape skin / seed lactic acid bacteria fermented product in Example 4. FIG. 5A is a graph showing the arthritis inhibitory action of type II collagen-induced mice of fermented grape skin and seed lactic acid bacteria in Example 5 over time. FIG. 5B is a graph showing the arthritis inhibitory action at the peak of type II collagen-induced mice of grape skin / seed lactic acid bacteria fermented product in Example 5. FIG. 6A is a graph showing the weight loss inhibitory effect of dextran sulfate sodium (DSS) -induced mice in grape skin / seed lactic acid bacteria fermentation product in Example 6. FIG. 6B is a graph showing the hematocrit level decrease-inhibiting effect of dextran sulfate sodium (DSS) -induced mice in grape skin / seed lactic acid bacteria fermented product in Example 6. FIG. 6C is a graph showing the action of inhibiting grape shortening in a dextran sulfate sodium (DSS) -induced mouse of a grape skin / seed lactic acid bacteria fermentation product in Example 6. FIG. 6D is a graph showing the TNF-α production inhibitory action of dextran sulfate sodium (DSS) -induced mice in the grape skin / seed lactic acid bacteria fermentation product in Example 6. FIG. 7A is a graph showing the passive skin anaphylaxis (PCA) inhibitory action of an acetone extract of a fermented grape skin / seed lactic acid bacterium or a residue thereof in Example 7. FIG. 7B shows passive skin anaphylaxis (PCA) of a hydrophilic-like substance (Fraction 1) and a hydrophobic-like substance (Fraction 2) fractionated by DIAION HP-20 from an acetone extract of a grape skin / seed lactic acid bacteria fermentation product in Example 7. ) It is a graph showing an inhibitory action. FIG. 7C shows substances (Fractions 3 to 5) that were fractionated by a difference in molecular weight with a Sephadex LH-20 column from the hydrophobic-like substance (Fraction 2) in the acetone extract of the grape skin / seed lactic acid bacteria fermentation product in Example 7. Is a TLC chromatogram when developed by thin layer chromatography (TLC). FIG. 7D shows the substances (Fractions 3 to 5) fractionated from the hydrophobic-like substance (Fraction 2) in the acetone extract of the grape skin / seed lactic acid bacteria fermented product in Example 7 using a Sephadex LH-20 column. Is a graph showing the passive skin anaphylaxis (PCA) inhibitory action of. FIG. 7E is a TLC chromatogram when an acetone extract of a grape skin / seed lactic acid bacteria fermentation product or an unfermented acetone extract of Example 7 is developed by thin layer chromatography (TLC). FIG. 7F shows the passive skin anaphylaxis (PCA) inhibitory action of a spot (spot e) specifically detected in a grape skin / seed lactic acid bacteria fermented product fractionated by thin layer chromatography (TLC) in Example 7. It is a graph.

(Medicine)
The medicament of the present invention is a medicament for treating or preventing at least one of allergic diseases and autoimmune diseases, and is obtained by fermenting at least one of grape skin and seed with lactic acid bacteria. Including fermented material, other components are included as necessary.

<Fermented grape skin and seed lactic acid bacteria>
The fermented grape skin / seed lactic acid bacteria is not particularly limited as long as it is obtained by fermenting at least one of grape skins and seeds (fruit skin / seed) with lactic acid bacteria, and is appropriately selected according to the purpose. Can do. In the present specification, the fermented grape skin / seed lactic acid bacteria may be referred to as “lactic acid bacteria fermented grape skin / seed” or the like.

-Grapes-
The grape is not particularly limited as long as it is a plant belonging to the Vitaceae grape genus (Vitis), and can be appropriately selected according to the purpose. For example, European grape (Vitis vinifera) ), American grapes (Vitis labrusca), Vitis amenrensis, Vitis coigniae, and hybrids thereof. The grape varieties are not particularly limited and may be appropriately selected depending on the purpose. For example, Koshu, Chardonnay, Muscat, Sauvignon Blanc, Semillon ( Semillon, Riesling, Silvaner, Muller-Thurgau, Kerner, Gewurztraminer, Chasselan, Chin Semi B ), Delaware, Bacchus, Pinot Gris, Pinot Plan (Pinot Blank), Fulmint (Furminnt), Muscadet, Muscadelle, Riesling, Cabernet Sauvignon, Muscat berry A (Muscat Ai b) Grenache, Merlot, Syrah, Negroamaro, Sangiovese, Tannat, Primitivo, Brunelet, Brunelet (Gamay), Concord (Co ncord, Sangiovese, Sansou (Cinsaut), Zinfandel, Seibel, Zweigeltrebe, Tempranillo, Barbera (Pirbar) Examples include Pinot Meunier, Petit Verdot, Malvasia, Malbec, Mourverder, and Kyoho.

-Skin and seed-
The skin and seeds are not particularly limited as long as they are at least one of the grape skins and seeds described above, and can be appropriately selected according to the purpose. It may be in a dry state. In addition, the size of the pericarp / seed may be a state in which at least one of the pericarp and seed is collected, or may be in a state of being cut into a desired size, or a fine powder ( It may be in a powdered state. Among them, the pericarp / seed is preferably in a dry state, and preferably in a fine powder state from the viewpoint of facilitating the action of lactic acid bacteria described later. That is, it is particularly preferable that the pericarp / seed is in a dry powder (dry powder) state. There is no restriction | limiting in particular as a method of producing the dry powder (dry powder) of the said skin and seed, According to the objective, it can select suitably, For example, it can carry out using a well-known method. There is no restriction | limiting in particular as an average particle diameter of the dry powder (dry powder) of the said skin and seed, Although it can select suitably according to the objective, For example, 10-200 mesh is preferable. When the average particle size is less than 10 mesh, the activity of the fermented product may be weak. On the other hand, when the average particle diameter is within the preferred range, it is advantageous in that a fermented product having strong activity can be obtained.
The grape skins and seeds are not limited in the method of obtaining them and can be appropriately selected according to the purpose. For example, the remaining grapes after squeezing the fruit juice in the production process of wine or juice. It is considered desirable to use firewood because it is cost effective and can reduce industrial waste.
Moreover, as the grape skin and seed, the grape skin may be used alone, the grape seed may be used alone, or the grape skin and seed may be used in combination. Also good.
Here, the raw material of the fermented product of the grape skin / seed lactic acid bacteria does not necessarily have to be composed of at least one of the grape skin and seed described above, and at least a part of the grape skin and seed described above. It is sufficient that at least one of them is included. For example, the grape skin / seed lactic acid bacteria fermented material may appropriately include the grape fruit, fruit juice, leaves, stems, etc. in addition to the grape skin / seed. There is no restriction | limiting in particular as usage-amount ratio with respect to the said fruit peel and seed, and the said grape peel and seed, It can select suitably according to the objective.

-Lactic acid bacteria-
The lactic acid bacterium is not particularly limited as long as it is a bacterium that produces lactic acid by fermentation, and can be appropriately selected according to the purpose. For example, Lactobacillus, Examples include bacteria belonging to the genus Bifidobacterium, the genus Lactococcus, the genus Leuconostoc and the genus Enterococcus. The seeds of the lactic acid bacteria are not particularly limited and may be appropriately selected according to the purpose. Among them, the seeds that can be efficiently propagated using the grape skins and seeds as a carbon source are used. It is preferable. Such preferred species include, for example, Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus salivalus (Lactobacillus salivalus), Lactobacillus s. (Lactobacillus kitatatonisi), Lactobacillus reuteri, Lactobacillus johnsonii, Lactobacillus acidophilus, Lactobacillus acidophilus, Lactobacillus brevis (Lactobacillus brevis), Lactobacillus Korinihorumisu (Lactobacillus coryniformis), Lactobacillus Karubatasu (Lactobacillus curvatus), Lactobacillus Deruburyukyi (Lactobacillus delbrueckii), Lactobacillus Fal'cie Minas (Lactobacillus farciminus), Lactobacillus rhamnosus (Lactobacillus rhamnosus); Bae As the genus Diococcus, Pediococcus damnosus, Pediococcus halophilus, Pediococcus parva Pediococcus parvulus, Pediococcus pentosaceus; Bifidobacterium, Bifidobacterium adolescentis, Bifidobacteria bifida Lactococcus cremoris, Lactococcus lactis; as the genus Leuconostoc, Leuconostoc oenos, Leuconostoc lactis, Leuconostoc lactis As a genus of Enterococcus, Enterococcus faecalis (Enterococcus faecalis) and the like can be mentioned. Among them, Lactobacillus plantarum is particularly preferable as a seed of the lactic acid bacterium because it efficiently proliferates using the grape peel / seed as a carbon source and the obtained fermented product has a strong activity.

-Fermentation-
The fermentation can be performed by fermenting a raw material containing at least a part of the above-described grape skin and seed using the lactic acid bacteria. There is no restriction | limiting in particular as the method of the said fermentation, According to the objective, it can select suitably, For example, the method etc. which add and culture | cultivate the said lactic acid bacteria to the said raw material are mentioned. More specifically, for example, the fermentation can be performed by the method described in Examples described later.

There is no restriction | limiting in particular as the usage-amount of the lactic acid bacteria at the time of the said fermentation, According to the objective, it can select suitably, For example, 10 < ¹² > CFU or more is preferable with respect to the said raw material 10g, and 10 < ¹³ > CFU or more is especially preferable. . If the amount of the lactic acid bacteria used is less than 10 ¹² CFU, the activity of the fermented product may be weakened. On the other hand, when the amount of the lactic acid bacterium used is within the particularly preferable range, it is advantageous in that a fermented product having strong activity can be obtained.

  There is no restriction | limiting in particular as temperature at the time of the said fermentation, According to the objective, it can select suitably, For example, 10-40 degreeC is preferable and 17-30 degreeC is especially preferable. If the temperature is less than 10 ° C, the growth of the bacteria may be deteriorated, and if it exceeds 40 ° C, the bacteria may not be able to grow. On the other hand, when the temperature is within the particularly preferable range, it is advantageous in that a fermented product having strong activity can be obtained.

  There is no restriction | limiting in particular as said fermentation time, According to the objective, it can select suitably, For example, 48-144 hours are preferable and 72 hours are especially preferable. When the fermentation time is less than 48 hours, the activity of the fermented product may be weak. On the other hand, when the fermentation time is within the particularly preferable range, it is advantageous in that a fermented product having strong activity can be obtained.

  By the fermentation, the grape skin / seed lactic acid bacteria fermented product can be obtained. The grape skin / seed lactic acid bacteria fermented product is used as it is or as an active ingredient of the pharmaceutical and the food of the present invention described later by appropriately processing.

-Extraction and purification-
The grape skin / seed lactic acid bacteria fermented product may be used in a further extracted and purified state. Further extraction and purification of the grape skin / seed lactic acid bacteria fermentation product is advantageous in that a more active substance can be obtained.

For example, as the grape skin / seed lactic acid bacteria fermentation product, an organic solvent extract obtained by further extracting the grape skin / seed lactic acid bacteria fermentation product with an organic solvent may be used. There is no restriction | limiting in particular as said organic solvent, Although it can select suitably according to the objective, For example, acetone, chloroform, methanol, ethanol, ethyl acetate, hexane etc. are mentioned, Among these, acetone is especially preferable.
Here, the amount of acetone used when extracting the fermented grape skin / seed lactic acid bacteria with acetone is not particularly limited and may be appropriately selected depending on the intended purpose. For example, the grape skin / seed The amount of 70% acetone is preferably 100 to 500 mL with respect to 1 g of lactic acid bacteria fermentation product.
Moreover, there is no restriction | limiting in particular as extraction temperature at the time of extracting the said grape skin and seed lactic-acid-bacteria fermented material with acetone, Although it can select suitably according to the objective, For example, 20-60 degreeC is preferable.
In addition, the extraction time when the grape skin / seed lactic acid bacteria fermented product is extracted with acetone is not particularly limited and may be appropriately selected depending on the intended purpose. For example, it is preferably 1 hour or longer, and 24 hours. The above is more preferable.
In addition, it is preferable to remove water | moisture content from the extract obtained by the said acetone extraction by freeze-drying, after removing acetone using a rotary evaporator etc.
The acetone extract of the grape skin / seed lactic acid bacteria fermented product is advantageous in that it has a strong activity and is particularly excellent in an inhibitory effect on vascular permeability enhancement.

Further, as the grape skin / seed lactic acid bacteria fermented product, a hydrophobic substance contained in the acetone extract of the grape skin / seed lactic acid bacteria fermented product may be used. Here, as the hydrophobic substance, when the acetone extract is fractionated into a hydrophilic-like substance and a hydrophobic-like substance by the method described in Example 7 to be described later, it is fractionated as a hydrophobic-like substance. It shall mean the substance to be used. In addition, the hydrophobic substance is not limited to the technique described in Example 7 to be described later, for example, by fractionating the acetone extract into a hydrophilic substance and a hydrophobic substance by a conventionally known technique, It can also be obtained.
The hydrophobic substance contained in the acetone extract of the grape skin / seed lactic acid bacteria fermented product is advantageous in that it has a strong activity and is particularly excellent in an inhibitory effect on vascular permeability enhancement.

The grape skin / seed lactic acid bacteria fermented product may be a hydrophobic substance contained in the acetone extract of the grape skin / seed lactic acid bacteria fermented product having a molecular weight of 100 to 1,000 by gel filtration chromatography. A substance that is The substance having a molecular weight in the range of 100 to 1,000 by the gel filtration chromatography can be obtained, for example, by fractionating the hydrophobic substance by gel filtration chromatography.
Here, the column used for the gel filtration chromatography is not particularly limited and may be appropriately selected depending on the purpose. For example, Sephadex LH-20 (GE Healthcare), Sephadex G20 (GE Healthcare), Bio-Gel P-1 (GE Healthcare) and the like can be mentioned, and among these, Sephadex LH-20 (GE Healthcare) is preferable.
The eluent used for the gel filtration chromatography is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include water, ethanol, a mixed solution of ethanol and water, and the like. Among these, a mixed liquid of ethanol and water is preferable.
Of the hydrophobic substances contained in the acetone extract of the grape skin / seed lactic acid bacteria fermented product, substances having a molecular weight in the range of 100 to 1,000 by gel filtration chromatography are highly active, and in particular, vascular permeability. This is advantageous in that it has an excellent suppression effect.

The grape skin / seed lactic acid bacteria fermented product is a hydrophobic substance contained in the acetone extract of the grape skin / seed lactic acid bacteria fermented product, and has a molecular weight of 100 to 1,000 by gel filtration chromatography. Among these substances, silica gel is used as an adsorbent, and when a chloroform-methanol mixed solvent having a volume ratio of chloroform to methanol of 15: 1 is used as a developing solvent, the Rf value in thin layer chromatography is 0. Substances within the range of 3 to 0.4 may be used.
Examples of the substance include an acetone extract of the fermented grape skin and seed lactic acid bacteria, and a hydrophobic substance having a molecular weight in the range of 100 to 1,000 by gel filtration chromatography contained in the acetone extract. Fractionation by thin layer chromatography under the conditions as described above (using silica gel as an adsorbent and a chloroform-methanol mixed solvent having a volume ratio of chloroform and methanol of 15: 1 as a developing solvent) Can be obtained. Here, it is preferable to use silica gel 60 (Merck) as the silica gel.
A thin layer under the above conditions among the hydrophobic substances contained in the acetone extract of the grape skin / seed lactic acid bacteria fermentation product and having a molecular weight in the range of 100 to 1,000 by gel filtration chromatography A substance having an Rf value in the range of 0.3 to 0.4 in chromatography is advantageous in that it has a strong activity and is particularly excellent in an inhibitory effect on vascular permeability enhancement. In addition, the substance is not found in unfermented grape skin / seed, and therefore, the substance is a substance newly produced by lactic acid bacteria fermentation of grape skin / seed and has the main activity in the fermented grape skin / seed lactic acid bacteria. It is considered to be an ingredient. (Note that this substance corresponds to the spot “e” in FIGS. 7C and 7E.)

  As the grape skin / seed lactic acid bacteria fermented product, by using the extracted / purified product at each stage as described above, it is possible to improve each action of the medicine, and particularly, to improve the vascular permeability enhancement action. It is advantageous in that it can be made.

-Content-
The content of the grape skin / seed lactic acid bacteria fermented product in the medicament is not particularly limited and can be appropriately selected according to the purpose. The medicament is the grape skin / seed lactic acid bacteria fermented product itself. May be.

<Other ingredients>
There is no restriction | limiting in particular as said other component, In the range which does not impair the effect of this invention, it can select suitably according to the objective, For example, a pharmacologically acceptable carrier etc. are mentioned. There is no restriction | limiting in particular as said carrier, For example, it can select suitably according to the pharmaceutical dosage form etc. which are mentioned later. Moreover, there is no restriction | limiting in particular also as content of the said other component in the said pharmaceutical, According to the objective, it can select suitably.

<Dosage form>
The pharmaceutical dosage form is not particularly limited, and can be appropriately selected according to a desired administration method. For example, oral solid preparations (tablets, coated tablets, granules, powders, capsules, etc.), Oral solutions (internal solutions, syrups, elixirs, etc.), injections (solutions, suspensions, solid preparations for erection, etc.), suppositories, ointments, patches, gels, creams, external powders, sprays And inhalable powders.

Examples of the oral solid preparation include, for example, the above-mentioned fermented grape skin and seed lactic acid bacteria, excipients, and additives such as binders, disintegrants, lubricants, colorants, flavoring and flavoring agents as necessary. And can be produced by a conventional method.
Examples of the excipient include lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, and silicic acid. Examples of the binder include water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl starch, methylcellulose, ethylcellulose, shellac, calcium phosphate, polyvinylpyrrolidone and the like. It is done. Examples of the disintegrant include dry starch, sodium alginate, agar powder, sodium bicarbonate, calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride, and lactose. Examples of the lubricant include purified talc, stearate, borax, and polyethylene glycol. Examples of the colorant include titanium oxide and iron oxide. Examples of the flavoring / flavoring agent include sucrose, orange peel, citric acid, tartaric acid and the like.

The oral solution can be produced by a conventional method, for example, by adding additives such as a taste-masking / flavoring agent, a buffering agent, and a stabilizer to the grape skin / seed lactic acid bacteria fermented product.
Examples of the flavoring / flavoring agent include sucrose, orange peel, citric acid, tartaric acid and the like. Examples of the buffer include sodium citrate. Examples of the stabilizer include tragacanth, gum arabic, and gelatin.

As the injection, for example, a pH adjuster, a buffer, a stabilizer, a tonicity agent, a local anesthetic, and the like are added to the grape skin / seed lactic acid bacteria fermented product. And intravenous injections can be produced.
Examples of the pH adjusting agent and the buffering agent include sodium citrate, sodium acetate, sodium phosphate and the like. Examples of the stabilizer include sodium pyrosulfite, EDTA, thioglycolic acid, thiolactic acid, and the like. Examples of the isotonic agent include sodium chloride and glucose. Examples of the local anesthetic include procaine hydrochloride and lidocaine hydrochloride.

  Examples of the suppository include a known suppository formulation carrier such as polyethylene glycol, lanolin, cacao butter, fatty acid triglyceride, and the like, and a Tween (TWEEN: registered trademark) as necessary. After adding a surfactant, etc., it can be produced by a conventional method.

The ointment can be produced, for example, by mixing a known base, stabilizer, wetting agent, preservative and the like into the grape skin / seed lactic acid bacteria fermented product and mixing them by a conventional method.
Examples of the base include liquid paraffin, white petrolatum, white beeswax, octyldodecyl alcohol, and paraffin. Examples of the preservative include methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, and the like.

  As the patch, for example, a cream, gel or paste as the ointment can be applied to a known support by a conventional method. Examples of the support include woven fabric, nonwoven fabric, soft vinyl chloride, polyethylene, polyurethane and other films made of cotton, suf, and chemical fibers, and foam sheets.

<Administration>
There is no restriction | limiting in particular as an administration object of the said pharmaceutical, According to the objective, it can select suitably, For example, a human, a mouse | mouth, a rat, a hamster, a dog, a cat, a cow, a horse, a pig, a monkey etc. are mentioned.
Moreover, there is no restriction | limiting in particular as the administration method of the said pharmaceutical, According to the dosage form etc. of the said pharmaceutical, it can select suitably, For example, oral administration, administration by injection, etc. are mentioned.
The dosage of the drug is not particularly limited and can be appropriately selected according to the age, weight, sex, symptom, etc. of the patient to be administered, and is an active ingredient per day for a human adult. The amount of the grape skin / seed lactic acid bacteria fermentation product is preferably about 50 mg to 5 g.
Further, the frequency of administration of the medicament is not particularly limited and can be appropriately selected according to the purpose. For example, the daily dose may be administered once a day, Multiple doses may be administered.
Moreover, there is no restriction | limiting in particular also as the administration time of the said pharmaceutical, According to the objective, it can select suitably, For example, the said pharmaceutical may be administered therapeutically or may be administered prophylactically. .

<Target>
The medicament is suitable for the treatment or prevention of at least one of allergic diseases and autoimmune diseases. Allergic diseases are diseases in which an excessive immune reaction occurs against antigens from the outside, and autoimmune diseases are diseases in which substances constituting the body of the body are recognized as antigens and an excessive immune reaction occurs. Commonly due to excessive immune response.
The “allergic disease” is not particularly limited and can be appropriately selected depending on the purpose. For example, as type I allergic disease, hay fever, allergic rhinitis, bronchial asthma, urticaria, atopic dermatitis, Anaphylactic shock, food allergy; type II allergic disease, autoimmune hemolytic anemia, pernicious anemia, rheumatic fever, Hashimoto disease; type III allergy, systemic lupus erythematosus, acute glomerulonephritis, rheumatoid arthritis, rheumatoid pneumonia, frequent occurrence Arteritis, allergic vasculitis; type IV allergy, contact dermatitis; The “autoimmune disease” is not particularly limited and can be appropriately selected according to the purpose. For example, as systemic autoimmune disease, rheumatoid arthritis, systemic lupus erythematosus, rheumatic fever, scleroderma, Sjogren's syndrome; organ-specific autoimmune diseases include inflammatory bowel diseases such as ulcerative colitis, autoimmune hemolytic anemia, type I diabetes, multiple sclerosis, myasthenia gravis, pernicious anemia . In addition, all the symptoms which may occur in relation to the allergic disease and the autoimmune disease can be subjects for treatment or prevention of the medicine.
Since the medicament contains at least the fermented product of grape skin and seed lactic acid bacteria, it has an excellent inhibitory effect on vascular permeability enhancement, an IgE antibody production inhibitory effect, an eosinophil infiltration inhibitory effect, an anti-inflammatory effect (for example, an edema inhibitory effect, a swelling) Suppressive action), TNF-α production inhibitory action, etc. Among the various diseases described above, it is considered to be particularly suitable for the treatment or prevention of type I allergic disease, rheumatoid arthritis, and inflammatory bowel disease. (See Examples, below).

(Food)
The food of the present invention is a food for improving or preventing at least one of allergic diseases and autoimmune diseases, and is obtained by fermenting at least one of grape skin and seed with lactic acid bacteria. Including fermented material, other components are included as necessary.

<Fermented grape skin and seed lactic acid bacteria>
The grape skin / seed lactic acid bacteria fermented product is obtained by fermenting at least one of grape skins and seeds (fruit skin / seed) with lactic acid bacteria, and the “grape”, “fruit skin / seed”, “lactic acid bacterium” The “fermentation” is as described in the above-mentioned item of the medicine of the present invention. The grape skin / seed lactic acid bacteria fermented product may be used in a further extracted and purified state. The “extraction / purification” is as described in the item of the pharmaceutical of the present invention.
The content of the grape skin / seed lactic acid bacteria fermented product in the food is not particularly limited and can be appropriately selected according to the purpose, and the food is the grape skin / seed lactic acid bacteria fermented product itself. May be.

<Other ingredients>
There is no restriction | limiting in particular as said other component, In the range which does not impair the effect of this invention, it can select suitably according to the objective, For example, various food raw materials etc. are mentioned. There is no restriction | limiting in particular as said food raw material, For example, it can select suitably according to the kind etc. of the said foodstuff mentioned later. Moreover, there is no restriction | limiting in particular also as content of the said other component in the said foodstuff, According to the objective, it can select suitably.

<Type of food>
The type of food is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include confectionery such as jelly, candy, chocolate, biscuit and gummy; Taste drinks such as tea, coffee and soft drinks; Dairy products such as raw milk, yogurt and ice cream; Vegetable and fruit processed products such as vegetable drinks, fruit drinks and jams; Liquid foods such as soups; Bread and noodles Processed cereals such as: various seasonings; processed meat and seafood such as hamburger, croquette, kamaboko, fried satsuma; and the like. There is no restriction | limiting in particular as a manufacturing method of these foodstuffs, For example, according to the manufacturing method of various normal foodstuffs, it can manufacture suitably.
Moreover, the said foodstuff may be manufactured as oral liquid preparations, such as oral solid preparations, such as a tablet, a granule, a capsule, and an internal use liquid preparation, a syrup preparation, for example. The method for producing the oral solid preparation and the oral liquid preparation is not particularly limited and can be appropriately selected depending on the purpose. it can.

The form of administration (intake) of the food is not particularly limited and may be appropriately selected depending on the purpose. For example, the form of administration of the medicament of the present invention (administration subject, administration method, dose, administration time) ) And can be administered (taken) as appropriate.
Moreover, there is no restriction | limiting in particular as the improvement or prevention object of the said food, It is the same as that of the treatment or prevention object of the pharmaceutical of the above-mentioned this invention, The said food is the improvement or at least one of allergic disease and an autoimmune disease. Suitable for prevention.

  Since the food contains at least the fermented product of grape skin and seed lactic acid bacteria, it has an excellent inhibitory effect on vascular permeability enhancement, an IgE antibody production inhibitory effect, an eosinophil infiltration inhibitory effect, an anti-inflammatory effect (for example, an edema inhibitory effect, a swelling) Inhibitory action), TNF-α production inhibitory action, etc. Therefore, the food is intended to improve or prevent allergic diseases such as type I allergic disease, rheumatoid arthritis, autoimmune diseases such as inflammatory bowel disease, etc. It is particularly useful as a functional food taken in

[effect]
ADVANTAGE OF THE INVENTION According to this invention, the pharmaceutical and foodstuff which utilized the grape peel and seed lactic-acid-bacteria fermented material which can treat, improve, and prevent allergic disease and autoimmune disease can be provided.
Since the medicine and food of the present invention can effectively use grape skins and seeds that have been discarded in the past, it can be said that they are excellent from the viewpoint of environment. The grape skin / seed lactic acid bacteria fermented product uses grapes which are natural foods. Therefore, the medicament and food of the present invention containing the grape skin / seed lactic acid bacteria fermented product are allergic diseases, autoimmune diseases. It can be said that it is not only excellent in the treatment, improvement, and prevention effects, but also extremely safe.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[Sample preparation method]
Preparation of the grape skin / seed lactic acid bacteria fermented product (lactic acid bacteria fermented grape skin / seed) in this example was performed by the following method, unless otherwise specified.
The grape skin and seeds of Koshu, Cabernet Sauvignon, Musca, Negroamaro, and Primitivo were pulverized to prepare respective grape skin / seed dry powders. As the mixed grape skin / seed dry powder, each grape skin / seed dry powder was mixed at a ratio of Koshu: Cabernet Sauvignon: Musca: Negro Amaro: Primivo = 3: 3: 1: 1: 1. After suspending in purified water such that the concentration of each grape skin / seed dry powder was 10% by mass, lactic acid bacteria (Lactobacillus plantarum (KU146)) were inoculated to a concentration of 1 × 10 ¹¹ CFU / ml. After culturing at 37 ° C. for 48 hours (2 days), the culture solution was collected and heat-treated at 80 ° C. for 2 hours. The obtained specimen was diluted with distilled water and adjusted to a desired concentration in each example.

(Example 1: Inhibition of passive skin anaphylaxis (PCA))
Anti-ovalbumin (OVA) IgE antibody-containing antiserum was subcutaneously administered to mice sensitized with local mast cells. By administering OVA antigen and inducing the antigen, antibody-sensitized mast cells It is known that degranulation is induced, and the inflammatory substance contained in the granules increases vascular permeability and causes local inflammation (passive skin anaphylaxis (PCA) reaction). Here, the substance capable of suppressing the PCA reaction by administration after mast cell antibody sensitization is considered to be an extremely useful substance for therapeutic administration, which is effective even after administration of type I allergic reaction. It is done. In Example 1, the therapeutic effect of the grape skin / seed lactic acid bacteria fermented product against such type I allergic reaction was confirmed as follows.
A 5-week-old male BALB / c mouse (Japan SLC Co., Ltd.) was passively sensitized by subcutaneously administering 0.01 ml of antiserum containing anti-OVA IgE antibody to the auricle. 24 hours after the sensitization, 0.1 mg / mouse of OVA and 0.625 mg / mouse of Evans blue dye (Wako Pure Chemical Industries, Ltd.) were intravenously administered to induce antigen. Thirty minutes after antigen induction, the ears were cut out and dissolved in 0.25 ml of 1N potassium hydroxide solution for 24 hours. After neutralizing with 0.125 ml of 3N phosphoric acid solution, 0.325 ml of acetone was added to extract the dye leaked to the sensitized site. After centrifuging at 2,500 rpm for 10 minutes, the acetone layer was recovered and the absorbance was measured at a wavelength of 595 nm to quantify the amount of dye. Each grape skin / seed lactic acid bacteria fermented product specimen was orally administered at a dose of 10 or 100 mg / kg 30 minutes before antigen induction. Further, as a comparative control, each untreated grape skin / seed powder or grape skin / seed powder inoculated with killed lactic acid bacteria was orally administered in the same manner at a dose of 100 mg / kg. Also, as a comparative control, only the amount of lactic acid bacteria contained in a fermented grape skin / seed lactic acid bacteria sample at a dose of 100 mg / kg was orally administered in the same manner. As a control, distilled water was orally administered in the same manner.
The results are shown in FIGS. 1A and 1B. The vertical axis shows the amount of dye leakage (% control) in each sample administration group when the amount of dye leakage in the control group administered with distilled water is 100%.
FIG. 1A shows the results of each grape skin / seed lactic acid bacteria fermented product. In the control group, antigen-induced inflammation occurred in the auricle and marked pigment leakage due to increased vascular permeability was observed. On the other hand, in the Koshu, Cabernet Sauvignon, and the mixed grape peel / seed lactic acid bacteria fermented product administration group, a dose-dependent inhibitory effect on pigment leakage was observed, and even at a dose of 10 mg / kg, compared to the control group, respectively. It was shown to significantly inhibit pigment leakage.
In addition, FIG. 1B shows the results other than the fermented grape skin and seed lactic acid bacteria (comparative control group). In the group to which 100 mg / kg dose of untreated grape skin / seed powder, unfermented product obtained by adding lactic acid bacteria killed to grape skin / seed powder, and a group containing only a considerable amount of lactic acid bacteria, all were significant compared to the control group No significant pigment leakage inhibitory effect was observed.
From these results, fermented grape skin and seed lactic acid bacteria suppress the increase in vascular permeability caused by degranulation of mast cells, and effectively suppress type I allergic reaction (immediate reaction) involving mast cells. It was shown to have the effect of On the other hand, no significant inhibitory action was observed in any sample administration group other than the grape skin / seed lactic acid bacteria fermented product, indicating that fermentation of grape skin / seed by lactic acid bacteria is important.

(Example 2: IgE antibody production inhibitory action)
By sensitizing a mouse by administering ovalbumin (OVA), sensitized Th2 cells are induced, and a large amount of IgE antibody is detected in the blood. It is known that eosinophils in the bronchi and alveoli are induced by administering the antigen to the airway and inducing the antigen to mice sensitized with antigen, and exhibit symptoms similar to asthma. In such an animal model, a substance that suppresses IgE antibody production and eosinophil infiltration into the bronchus and alveoli by administration from the time of antigen sensitization prevents type I allergic reaction induced by Th2 cells. It can be suppressed. In Example 2 and Example 3 to be described later, the preventive effect of the grape skin / seed lactic acid bacteria fermented product against such type I allergic reaction was confirmed as follows.
5-week-old male BALB / c mice (Japan SLC) were injected intraperitoneally with ovalbumin (OVA; Sigma) 0.1 mg / mouse and aluminum hydroxide gel 1.6 mg / mouse every other week. The sensitization was carried out by administration, and two weeks after the first sensitization, 0.1 mg / mouse of OVA was administered nasally every other day for antigen induction. One day after the final antigen induction, blood was collected from the fundus, centrifuged at 2500 rpm for 10 minutes, and serum was separated. The antigen-specific IgE antibody titer in serum was measured with an ELISA kit (Dainippon Sumitomo Pharma Co., Ltd.). Each sample was orally administered at a dose of 100 mg / kg once a day from the first antigen sensitization day to the final antigen induction day, a total of 13 times, and distilled water was orally administered to the control group in the same manner.
The results are shown in FIG. In the control group, antigen-specific IgE antibody production was induced by antigen sensitization, whereas in the grape skin / seed lactic acid bacteria fermented product administration group, a significant decrease in IgE amount was observed compared to the control group. . From this, it was shown that the grape skin / seed lactic acid bacteria fermented product has an action of suppressing the production of IgE antibody and effectively suppressing the type I allergic reaction involving the IgE antibody. On the other hand, in the sample administration groups other than the grape skin / seed lactic acid bacteria fermented product, no significant inhibitory action was observed, indicating that the fermentation of grape skin / seed with lactic acid bacteria is important.

(Example 3: Inhibition of eosinophil infiltration into bronchi and alveoli)
In the same manner as in Example 2, 1 ml of phosphate buffered saline (PBS) was injected into the antigen-sensitized and antigen-induced mice through the respiratory tract and bronchial and alveolar lavage fluids were collected. After measuring the total number of cells contained in the bronchial and alveolar lavage fluid, a smear was prepared, and Giemsa staining was performed to measure the ratio of eosinophils.
The results are shown in FIG. In the control group, eosinophil infiltration into the bronchus and alveoli was observed due to antigen sensitization and induction, but almost none was observed in the grape skin / seed lactic acid bacteria fermented group. From this, it was shown that fermented grape skin and seed lactic acid bacteria have an action of effectively suppressing type I allergic reaction (late reaction) involving eosinophils. On the other hand, in the untreated grape skin / seed powder administration group, such an inhibitory effect was not observed, indicating that fermentation of grape skin / seed with lactic acid bacteria is important.

(Example 4: Inhibition of foot edema in carrageenan-induced mice)
It is known that when carrageenan, one of the inflammation-inducing agents, is subcutaneously administered to the footpad of mice, carrageenan is directly taken up by local macrophages and activates macrophages. Paw edema is caused by inflammatory substances such as TNF-α, IL-1, IL-6, PGE2, and NO produced from activated macrophages. This substance that suppresses inflammation is considered to be effective against autoimmune diseases such as rheumatoid arthritis induced by macrophages as effector cells. In Example 4, the inhibitory effect of the grape skin / seed lactic acid bacteria fermented product on the paw edema of such carrageenan-induced mice was confirmed as follows.
Seven-week-old male ICR mice (Japan SLC Co., Ltd.) were intradermally administered with 0.025 ml of 2% carrageenan (Wako Pure Chemical Industries, Ltd.) solution to the right hind footpad to induce paw edema. The foot thickness was measured using a dial thickness gauge at 1, 3, and 5 hours after carrageenan administration. Each specimen was orally administered at a dose of 100 mg / kg 1 hour before carrageenan administration, and distilled water was orally administered to the control group in the same manner. The determination of foot edema was calculated by the following formula.
Formula: “Increase rate of foot edema = (foot thickness after carrageenan administration−foot thickness before carrageenan administration) / foot thickness before carrageenan administration”
The results are shown in FIG. For the foot edema induced by carrageenan administration, Koshu, Cabernet Sauvignon, and the mixed grape peel / seed lactic acid bacteria fermented product administration group all showed significant edema-inhibiting action. From this, it was shown that the fermented grape skin and seed lactic acid bacteria have an action of effectively suppressing non-specific immediate inflammation caused by macrophages.

(Example 5: Arthritis inhibitory effect of type II collagen-induced mice)
Type II collagen is a major component of joints, and it is known that when mice are forcibly immunized with type II collagen, an autoimmune state occurs and arthritis develops. This is considered to be a model of rheumatoid arthritis. Antigen-specific Th1 cells for type II collagen are induced, macrophages are activated by Th1 cells, and TNF-α, IL- produced from the activated macrophages. 1. Inflammatory substances such as IL-6, PGE2 and NO cause arthritis (J. Exp. Med. 1997; 146: 857-68). This substance that suppresses arthritis is considered to be effective against autoimmune diseases such as rheumatoid arthritis. In Example 5, the inhibitory effect of fermented grape skin / seed lactic acid bacteria on arthritis in type II collagen-induced mice was confirmed as follows.
Chicken-derived type II collagen (Sigma Aldrich) was dissolved in a 0.1 mM acetic acid solution and adjusted to a concentration of 2 mg / ml. An equal amount of complete Freund's adjuvant (Gibco) was mixed with this solution to prepare an emulsion. 0.1 ml of the emulsion was sensitized by intradermal administration into the tail base skin of male DBA / 1J mice (Japan SLC Co., Ltd.) 9 weeks old, and secondary sensitization was performed in the same manner 21 days later. Each sample was orally administered daily at a dose of 150 mg / kg after the second sensitization of the antigen, and distilled water was orally administered to the control group in the same manner. The degree of swelling of the front and rear legs was observed, and a clinical score was assigned according to the following evaluation.
[Clinical score]
Score 0: no swelling.
Score 1: Redness is seen in palms, heels, fingertips, etc.
Score 2: Swelling is observed on a part of the limbs.
Score 3: swelling is observed in the entire limb.
Score 4: Strong swelling is observed in the entire limbs, and surface roughness and inflammation are observed.
Score 5: Significant swelling is seen in the entire limb, and deformation is seen in the joint.
Each of the four legs was observed, and the total was shown as a clinical score for one individual.
The results are shown in FIGS. 5A and 5B. FIG. 5A shows the daily change of the clinical score, and FIG. 5B shows the value of the clinical score at the peak. In the control group, the score increased rapidly after the secondary sensitization, and the peak score was 14.3. On the other hand, in each grape skin / seed lactic acid bacteria fermented product administration group, an effect of suppressing the onset of arthritis was recognized from around 28 days after sensitization, and Koshu, Cabernet Sauvignon, and mixed grape skin / seed. Regarding the scores at the peak of the lactic acid bacteria fermented product administration group, 9.3, 12.2, and 11.3 were all observed, and a significant effect of suppressing the onset of arthritis was observed in all. From this, it was shown that fermented grape skin and seed lactic acid bacteria are effective against autoimmune diseases such as rheumatoid arthritis.

(Example 6: Inhibitory effect on ulcerative colitis-related symptoms in dextran sulfate sodium (DSS) -induced mice)
It is known that dextran sulfate sodium (DSS), which is one of the sulfate polysaccharides, can be freely consumed by mice to create a pathological model similar to ulcerative colitis, which is frequently used as an experimental ulcerative colitis model Has been. Substances that suppress ulcerative colitis-related symptoms in this model are considered effective against inflammatory bowel diseases such as ulcerative colitis. In Example 6, the inhibitory effect of fermented grape skin and seed lactic acid bacteria on ulcerative colitis-related symptoms in such dextran sulfate sodium (DSS) -induced mice was confirmed as follows.
Seven-week old male BALB / c mice (Japan SLC Co., Ltd.) were allowed to drink 5% dextran sulfate sodium (DSS; Wako Pure Chemical Industries, Ltd.) in a water bottle. One week after the start of drinking, the mice were weighed, whole blood was collected from the heart, and the large intestine was removed. For blood, the hematocrit value was measured with a blood cell counter (Coulter Counter; Coulter Company), and the length of the large intestine from the lower cecum to the anus was measured. Further, 1 ml of a phosphate buffer containing 100 nM phenylmethylsulfonyl fluoride (PMSF; Wako Pure Chemical Industries, Ltd.) is added to each large intestine and pulverized. After centrifugation at 15,000 rpm for 10 minutes, TNF contained in the supernatant is added. -The amount of α was measured using an ELISA kit (BD). Koshu grape peel / seed lactic acid bacteria fermented product was orally administered at a dose of 100 mg / kg once a day from the start of DSS drinking, a total of 7 times. As a comparative control, untreated (unfermented) Koshu grape peel / seed powder was orally administered at a dose of 100 mg / kg. As a control, the same amount of purified water was administered. The values in the graph show the average value and standard error of 5 animals per group.
The results are shown in FIGS. 6A to 6D. In the control group, when DSS was allowed to drink freely in mice, symptoms such as diarrhea, bloody stool, weight loss, etc. were observed one week after the start of drinking. Shortening and a decrease in the hematocrit value were also observed, confirming the symptoms of ulcerative colitis.
FIG. 6A shows the inhibitory effect on weight loss. The graph shows the body weight after administration when the body weight before administration of DSS was taken as 100%, and a decrease in body weight was observed in the control group one week after the start of drinking due to free drinking of DSS. On the other hand, a significant recovery effect on body weight loss was observed in the group of mice orally administered with grape skin and seed lactic acid bacteria fermented product. This significant recovery effect was not observed when unfermented (untreated) grape skin / seed powder was administered.
FIG. 6B shows the inhibitory effect on the decrease in hematocrit value. In the control group, a decrease in hematocrit value was observed, but in the group of mice that were orally administered with grape skin / fermented lactic acid bacteria, a significant recovery effect on hematocrit value was observed. This significant recovery effect was not observed when unfermented (untreated) grape skin / seed powder was administered.
FIG. 6C shows the inhibitory effect on shortening of the large intestine. Shortening of the large intestine was observed in the control group, but a significant recovery effect was observed in the group of mice orally administered with the grape skin and fermented lactic acid bacteria. This significant recovery effect was not observed when unfermented (untreated) grape skin / seed powder was administered.
FIG. 6D shows the inhibitory action of TNF-α in large intestine homogenate. In the control group, increased production of TNF-α in the large intestine homogenate was observed, but a significant inhibitory effect was observed in the group of mice orally administered with grape skin / fermented lactic acid bacteria. This significant inhibitory effect was not observed with the treatment of untreated (unfermented) grape skin and seed powder.
Based on these results, fermented grape skin and seed lactic acid bacteria are prone to ulcerative colitis such as increased production of TNF-α, which is thought to be important for the development of ulcerative colitis, weight loss, decreased hematocrit value, and shortened colon. It was shown to have the effect of suppressing the symptoms. From these results, it was shown that the fermented grape skin and seed lactic acid bacteria are effective for the treatment, improvement, or prevention of autoimmune diseases including inflammatory bowel diseases such as ulcerative colitis. Moreover, it was shown that such an action was not observed in untreated (unfermented) grape skin / seed powder, but was expressed by lactic acid bacteria fermentation of grape skin / seed.

(Example 7: Inhibition of passive skin anaphylaxis (PCA) by extraction / purified product from grape skin and seed lactic acid bacteria fermented product)
In this Example 7, the passive skin anaphylaxis (PCA) inhibitory action of the extract / purified product from the grape skin / fermented lactic acid bacteria was confirmed. Here, the passive skin anaphylaxis (PCA) inhibitory action of each extract / purified product was evaluated by the same method as in Example 1. The results of the passive skin anaphylaxis (PCA) inhibitory action of each extract / purified product are shown in FIGS. 7A, 7B, 7D, and 7F. In each case, the vertical axis indicates the amount of pigment leakage in the control group administered with distilled water. The amount of pigment leakage (% control) in each extract / purified product administration group was shown.
100 g of 70% acetone was added to 5 g of Koshu grape peel and seed lactic acid bacteria fermented product, and sonication was performed at room temperature for 1 hour to extract components. The recovered extract was evaporated to dryness on a rotary vacuum evaporator and freeze-dried to obtain an acetone extract component. As a result, 0.9 g of acetone extract was obtained from 5 g of Koshu grape peel / seed lactic acid bacteria fermentation product. FIG. 7A shows the results of examining the effect of oral administration of an equivalent amount of 300 mg / kg of Koshu grape peel / seed lactic acid bacteria fermented product on the PCA reaction inhibitory action of the acetone extract and its residue. Although a strong inhibitory action was observed in the acetone extract, but not in the residue, it was suggested that the components involved in PCA reaction inhibition were extracted by the acetone extraction.
Subsequently, fractionation operation was further performed from the acetone extract. First, it was largely divided into a hydrophilic-like substance and a hydrophobic-like substance. 1 g of acetone extract was dissolved in 100 ml of methanol, mixed with 2 g of synthetic adsorbent aromatic beads DIAION HP-20 (NIPPON RENSUI), and methanol was adsorbed by distillation using an evaporator. The DIAION HP-20 adsorbed with the extract was layered on top of the DIAION HP-20 packed in advance in the column, and the flowed out by distilled water as the eluent was adsorbed to Fraction 1 and DIAION HP-20, The one that flowed out with 100% methanol was designated as Fraction 2. That is, Fraction 1 and Fraction 2 were obtained as a hydrophilic-like substance and a hydrophobic-like substance, and the recoveries were 0.58 g and 0.39 g, respectively. The PCA reaction inhibitory action of Fraction 1 and Fraction 2 is shown in FIG. 7B. Fraction 1 did not show an inhibitory effect at a dose of 100 mg / kg, but Fraction 2 showed a significant inhibitory effect. From this, it was found that a compound biased to hydrophobicity contained in the acetone extract is important for PCA reaction inhibition.
Next, the faction 2 showing a significant inhibitory action in the PCA reaction was further fractionated by the difference in molecular weight. That is, 0.1 g of Fraction 2 dissolved in 1 ml of methanol was added to a column packed with 40 ml of swollen Sephadex LH-20 resin (GE Healthcare) and then eluted with 200 ml of 70% ethanol. The first 40 ml eluted is discarded, and the next 40 m are collected and fraction 3 (molecular weight 100-200), Fraction 4 (molecular weight 200-300), Fraction 5 (molecular weight 300-1,000) in the order of elution. ). The recovery amount of each Fraction was obtained in the weight of Fraction 3: 0.014 g, Fraction 4: 0.033 g, Fraction 5: 0.047 g. FIG. 7C shows the component search for each fraction by TLC [adsorbent: silica gel 60 (particle size: 0.040 to 0.063 mm), developing solvent: chloroform: methanol = 15: 1, color reagent: 10% sulfuric acid]. Results are shown. Eight spots indicated by a to h were confirmed. Here, the Rf value of the spot e under the above conditions was 0.38. Moreover, the result of the PCA reaction inhibitory effect of Fraction3, Fraction4, and Fraction5 is shown in FIG. 7D. Fraction 3 and Fraction 4 did not show an inhibitory action at a dose of 100 mg / kg, whereas Fraction 5 showed a significant inhibitory action. It was suggested that the substance exhibiting PCA reaction inhibitory activity may be any or all of the spots e, f, and g detected from Fraction 5.
FIG. 7E shows a TLC [adsorbent; silica gel 60 (particle size: 0.040) in 70% acetone extract component of Koshu grape skin / seed lactic acid bacteria fermented product and unfermented product (untreated Koshu grape skin / seed powder). To 0.063 mm), developing solvent; chloroform: methanol: ethyl acetate = 15: 1: 1 (volume ratio), color reagent; 10% sulfuric acid]. Of the eight spots a to h recognized in the lactic acid bacteria fermented product, spot e is not confirmed in the unfermented product, and this substance may be a substance involved in the suppression of PCA reaction newly generated by lactic acid bacteria fermentation. Sex was suggested. Here, the Rf value of the spot e under the above conditions was 0.47.
In order to isolate this spot e, 100 mg of 70% acetone extract of Koshu grape peel / seed lactic acid bacteria fermented product was dissolved in a methanol-chloroform mixed solvent (methanol: chloroform = 2: 1 (volume ratio)) and used for fractionation. Development was performed on a TLC plate (silica gel 60; Merck). As a developing solvent, chloroform: methanol: ethyl acetate = 15: 1: 1 (volume ratio) was used. Here, the Rf value of the spot e under the above conditions was 0.47. The silica gel spot e was peeled off from the TLC plate, and ethyl acetate was added to the peeled powder to dissolve only the extract. The silica gel was removed from this solution by cotton plug filtration, and 0.43 mg of an extract of spot e was obtained. FIG. 7F shows the PCA reaction inhibitory action of this substance. In contrast to the inhibitory effect at 10 mg / kg dose of fermented grape skin and seed lactic acid bacteria, spot e, which is an extract / purified product, has a stronger inhibitory effect even at a lower dose of 3 mg / kg. An increase in specific activity was shown. Based on these results, the substance extracted and purified from the fermented grape skin and seed lactic acid bacteria is a substance newly produced by lactic acid bacteria fermentation and is a substance mainly involved in the suppression of type I allergic reaction. Was suggested.

  The medicament and food of the present invention using a grape skin / seed lactic acid bacteria fermented product, for example, for treatment, improvement or prevention of allergic diseases such as type I allergic diseases, rheumatoid arthritis, autoimmune diseases such as inflammatory bowel diseases, It can be suitably used.

Claims (18)

  A medicament for treating or preventing at least one of allergic diseases and autoimmune diseases, comprising a grape skin / seed lactic acid bacteria fermented product obtained by fermenting at least one of grape skin and seed with lactic acid bacteria A medicinal drug.   The pharmaceutical according to claim 1, wherein the grape skin / seed lactic acid bacteria fermented product is an organic solvent extract obtained by further extracting the grape skin / seed lactic acid bacteria fermented product with an organic solvent.   The medicament according to any one of claims 1 to 2, wherein the fermented grape skin / seed lactic acid bacteria is an acetone extract obtained by further extracting the fermented grape skin / seed lactic acid bacteria with acetone.   The grape skin / seed lactic acid bacteria fermented product is a hydrophobic substance contained in an acetone extract obtained by further extracting the grape skin / seed lactic acid bacteria fermented product with acetone. Medicine.   Grape peel / seed lactic acid bacteria fermented product is a hydrophobic substance contained in an acetone extract obtained by further extracting the grape skin / seed lactic acid bacteria fermented product with acetone, and gel filtration chromatography of the hydrophobic substance The pharmaceutical according to any one of claims 1 to 4, wherein the molecular weight is in the range of 100 to 1,000.   Grape peel / seed lactic acid bacteria fermented product is a hydrophobic substance contained in an acetone extract obtained by further extracting the grape skin / seed lactic acid bacteria fermented product with acetone, and gel filtration chromatography of the hydrophobic substance Thin film when using a chloroform-methanol mixed solvent having a molecular weight in the range of 100 to 1,000, using silica gel as an adsorbent, and having a volume ratio of chloroform to methanol of 15: 1 as a developing solvent. The pharmaceutical according to any one of claims 1 to 5, wherein the Rf value in layer chromatography is in the range of 0.3 to 0.4.   The medicament according to any one of claims 1 to 6, which is used for treatment or prevention of type I allergic disease.   The medicament according to any one of claims 1 to 6, which is used for treatment or prevention of rheumatoid arthritis.   The medicament according to any one of claims 1 to 6, which is used for treatment or prevention of inflammatory bowel disease.   A food for improving or preventing at least one of allergic diseases and autoimmune diseases, comprising a grape skin / seed lactic acid bacteria fermented product obtained by fermenting at least one of grape skin and seed with lactic acid bacteria Characteristic food.   The food product according to claim 10, wherein the grape skin / seed lactic acid bacteria fermented product is an organic solvent extract obtained by further extracting the grape skin / seed lactic acid bacteria fermented product with an organic solvent.   The food product according to any one of claims 10 to 11, wherein the fermented grape skin / seed lactic acid bacteria is an acetone extract obtained by further extracting the fermented grape skin / seed lactic acid bacteria with acetone.   The grape skin / seed lactic acid bacteria fermented product is a hydrophobic substance contained in an acetone extract obtained by further extracting the grape skin / seed lactic acid bacteria fermented product with acetone. Food.   Grape peel / seed lactic acid bacteria fermented product is a hydrophobic substance contained in an acetone extract obtained by further extracting the grape skin / seed lactic acid bacteria fermented product with acetone, and gel filtration chromatography of the hydrophobic substance The food according to any one of claims 10 to 13, wherein the molecular weight is in the range of 100 to 1,000.   Grape peel / seed lactic acid bacteria fermented product is a hydrophobic substance contained in an acetone extract obtained by further extracting the grape skin / seed lactic acid bacteria fermented product with acetone, and gel filtration chromatography of the hydrophobic substance Thin film when using a chloroform-methanol mixed solvent having a molecular weight in the range of 100 to 1,000, using silica gel as an adsorbent, and having a volume ratio of chloroform to methanol of 15: 1 as a developing solvent. The food according to any one of claims 10 to 14, wherein the Rf value in layer chromatography is in the range of 0.3 to 0.4.   The food according to any one of claims 10 to 15, which is used for improvement or prevention of a type I allergic disease.   The food according to any one of claims 10 to 15, which is used for improving or preventing rheumatoid arthritis.   The food according to any one of claims 10 to 15, which is used for improvement or prevention of inflammatory bowel disease.