JP2006241023A - Agent for inducing expression or accelerating production of antibacterial peptide in digestive tract tissue - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a pharmaceutical given by searching a component having action of inducing expression or accelerating production of antibacterial peptide defensin, containing the component as an active ingredient, and therefore having the action of inducing the expression or accelerating the production of the antibacterial peptide defensin, and to provide food and drink having the action. <P>SOLUTION: The agent for inducing the expression or accelerating the production of the antibacterial peptide in a digestive tract tissue contains a mannan-containing component derived from yeast as the active ingredient. The mannan-containing component preferably has a mannose content of ≥60 mass%. The agent is used for inducing the expression or accelerating the production of α-defensin or β-defensin as the antibacterial peptide. The pharmaceutical and the food and drink contain the agent, respectively. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to an expression induction or production promoter for an antibacterial peptide containing a yeast-derived mannan-containing component as an active ingredient, and a pharmaceutical or a food or drink containing the same.

  Antibacterial peptides are small molecules of 10 to 50 amino acid residues that can kill microorganisms such as bacteria, fungi, and viruses by passing through cell membranes. Since antimicrobial peptides were discovered in arthropods in 1981, about 500 molecules have been identified in higher organisms such as plants, insects and mammals.

  In animals, it has been found in Drosophila, bovine neutrophils, leukocytes, pig small intestine, frogs, scorpions, bees, spiders and the like. In plants, it is widely found in Brassicaceae, Gramineae, etc., and is said to play a role in protecting seeds and nutrient tissues from bacteria. These antimicrobial peptides vary considerably in size and structure, but are generally membrane-active amphiphilic molecules with a positive charge at neutral pH (Zalsoff M, Antimicrobial peptides s of multicellular organisms. Nature 2002; 415 ; 389-395). The majority of antimicrobial peptides are found in animal epithelial tissues and play an important role in biodefense as the first immune barrier. It has also been found in human gastrointestinal and digestive systems, skin, mucous membranes and mouse skin and gastrointestinal tract.

  Defensins, which are a kind of such antibacterial peptides, have a common structure having six cysteines constituting three intramolecular silsulfide bonds. There are two main types of defensin: α-defensin (present in neutrophils and pancreatic cells of the small intestine crypt) and β-defensin (longer than α-defensins, present in epithelial cells such as mucous membranes, skin, and trachea) ).

  Among them, α-defensin (cryptodine) secreted from small intestinal crypt paneth cells has an extremely strong bactericidal ability, and plays an important role in intestinal immunity by acting on bactericidal action against bacteria and viruses. It is possible to exert an infection protection function against bacteria or viruses.

  Therefore, if there is a highly safe and non-toxic substance that has the function of promoting the production and secretion of antibacterial peptides from pancreatic cells of the intestinal epithelium, the immune stimulating activity of the living body is increased, and diseases caused by bacterial and viral infections It is thought that it can be an effective therapeutic agent for the disease.

  In addition, β-defensins that are inductively expressed in the skin, trachea, lungs, mucous membranes, etc. also play an important role in exerting defense and immune functions at sites directly in contact with the outside world. Substances that promote expression are also considered to be effective as therapeutic agents for diseases caused by antibacterial agents and viruses.

The following examples are known for such induction and enhancement of defensin expression.
(1) Yeast-derived polysaccharide-containing composition and production method thereof (Japanese Patent Laid-Open No. 2003-197 (Patent Document 1), expression induction of defensin gene by yeast-containing composition containing a large amount of β-glucan Cell)
(2) Mammal antibacterial peptide expression inducing and / or enhancing composition, food and drink, pharmaceutical and quasi-drug containing the composition (Japanese Patent Laid-Open No. 2003-262 (Patent Document 2), yeast-derived insoluble fraction Induction of antibacterial peptide defensin gene expression by human epidermal keratinocytes).
In any of the examples, an insoluble component obtained by degrading yeast with an enzyme is described as an active component.

  By the way, yeasts can be classified into insoluble components (cell walls) obtained by enzymatic degradation and yeast extracts that are soluble components. Furthermore, the cell wall is mainly composed of polysaccharides such as glucan, mannan and chitin, and can be classified into an insoluble component (polysaccharide) and a soluble component (polysaccharide) by dissolving the cell wall with an alkali. The alkali-insoluble component is mainly insoluble glucan, and the alkali-soluble component includes mannan, soluble glucan and denatured protein.

Mannan, a cell wall of yeast and an alkali-soluble component (polysaccharide), is known to have an effect of preventing infection of food animals with Salmonella and the like. An example is shown below.
(1) Infectious disease prevention feed composition (Japanese Patent Laid-Open No. 2001-8636 (Patent Document 3), prevention of infectious diseases caused by betaine and dried beer yeast cell wall),
(2) Feed for preventing contamination with Salmonella (Japanese Patent Laid-Open No. 2000-116338 (Patent Document 4), prevention of Salmonella colonization with mannose and galactose),
(3) Livestock and poultry feed containing mannan derived from microorganisms (Japanese Patent Laid-Open No. 9-84529 (Patent Document 5), gram-negative bacteria excretion effect by microorganism-derived mannan and yeast cell wall),
(4) Mannose polysaccharide-containing feed (Japanese Patent Laid-Open No. 8-173055 (Patent Document 6), Salmonella contamination prevention by mannose polysaccharide-containing feed),
(5) Feed for preventing infection of harmful bacteria (Japanese Unexamined Patent Publication No. 8-38064 (Patent Document 7), prevention of harmful bacterial infections by mannoses, etc.)

Furthermore, it is known that mannan which is an alkali-soluble component (polysaccharide) of the yeast cell wall has an allergic constitution improving effect (Japanese Patent Laid-Open No. 2004-224772 (Patent Document 8)). Furthermore, it is also known that both the alkali-insoluble component and the soluble component of the yeast cell wall have a gastrointestinal immunity stimulating effect (Japanese Patent Laid-Open No. 2004-107281 (Patent Document 9)).
Japanese Patent Laid-Open No. 2003-197 JP 2003-262 A JP 2001-8636 A JP 2000-116338 A JP 9-84529 A JP-A-8-173055 JP-A-8-38064 Japanese Patent Application Laid-Open No. 2004-224772 JP 2004-107281 A

  Patent Documents 1 and 2 describe β-glucan as an active ingredient for inducing and enhancing defensin expression among insoluble components obtained by degrading yeast with an enzyme. However, the insoluble components of the yeast cell wall may have a bitter taste, a unique odor, and may be colored, and have properties such as low water solubility and poor dispersibility in water. Therefore, oral administration is difficult as it is, and application to food and drink is limited. In Patent Documents 1 and 2, the expression of the defensin gene is performed only in an in vitro cultured cell system, and evaluation in a mouse animal experiment close to an actual human form has not been performed.

  Patent Documents 3 to 7 only disclose that yeast cell walls and mannan have an effect of preventing infection of edible animals such as Salmonella, but do not mention expression induction or promotion of production of antibacterial peptides including defensin. .

  Patent document 8 only describes the allergic constitution improving effect of mannan, and does not mention expression induction or production promotion of antibacterial peptide. Patent document 9 describes that the cell wall and insoluble and soluble components of brewer's yeast have an intestinal IgA production enhancing effect, but does not mention the induction or promotion of production of antibacterial peptides.

  If it is possible to find a component having the action of promoting the expression induction or production of the antibacterial peptide defensin as described above from non-toxic and safe naturally-derived materials, it is possible to enhance the immunostimulatory ability of the living body, It is considered possible to develop pharmaceuticals and foods and drinks that can prevent AIDS onset. As described above, Patent Documents 1 and 2 disclose that β-glucan, which is an insoluble component obtained by degrading yeast with an enzyme, has an action of inducing and enhancing the expression of the antibacterial peptide defensin. Certain β-glucans also have the problems described above.

  Therefore, the present invention searches for a component having an action of promoting expression induction or production of the antibacterial peptide defensin, and a pharmaceutical and a food and drink having an action of promoting expression induction or production of the antibacterial peptide defensin containing the same as an active ingredient Is to provide.

  In addition to the above-mentioned patent documents, many reports on the immunostimulatory action of food materials have been made so far. An immunity improving action has also been found for mannan produced from the yeast cell wall fraction (Patent Document 9). However, the detailed mechanism of this immunity improving action has not been clarified, and there is only knowledge about acquired immunity (or adaptive immunity) involving antibodies and the like. Does not mention anything. The present inventors have found the function of promoting the expression induction and production of defensin, an antibacterial peptide, to the digestive tract in which the mannan-containing component derived from yeast plays an important role in intestinal immunity, and completed the present invention. .

The present invention for solving the above problems is as follows.
[Claim 1] An agent for inducing expression or promoting production of antibacterial peptides in digestive tract tissues, comprising a mannan-containing component derived from yeast as an active ingredient.
[2] The accelerator according to [1], wherein the mannan-containing component has a mannose content of 60% by mass or more.
[3] The promoter according to [1] or [2], wherein the mannan-containing component is obtained from a yeast cell or a culture solution.
[Claim 4] The promoter according to any one of claims 1 to 3, wherein the yeast is a brewery yeast.
[Claim 5] The accelerator according to any one of claims 1 to 4, wherein the accelerator is water-soluble, and the aqueous solution of the accelerator has low viscosity.
[6] The accelerator according to [5], which is liquid and contains 100 mg to 2 g of mannose per 100 mL.
[7] The promoter according to [1], which is used for inducing expression or promoting production of α-defensin or β-defensin as the antimicrobial peptide.
[8] The promoter according to any one of [1] to [7], which has an immunostimulatory effect, an infectious disease preventive effect and / or an AIDS onset suppressing effect.
[Claim 9] A pharmaceutical or food or drink comprising the promoter according to any one of claims 1 to 8.

  According to the present invention, as an antibacterial peptide, a substance effective for inducing expression or promoting production of α-defensin or β-defensin can be provided, whereby an immunostimulatory action, an infectious disease preventive action, and / or an AIDS onset inhibitory action can be provided. Is expected to have Furthermore, this substance can be easily used as a material for pharmaceuticals and foods and drinks.

  The present invention relates to an antibacterial peptide expression inducing or production promoter (hereinafter, sometimes simply referred to as the promoter of the present invention) in digestive tract tissues, which comprises a yeast-derived mannan-containing component as an active ingredient.

  The promoter of the present invention has a yeast-derived mannan-containing component as an active ingredient, and the yeast-derived mannan-containing component has a mannose content of 60% by mass or more. It is preferable from the viewpoint of high expression induction or production promotion effect. The yeast cell wall contains about 10-30% mannan, and the mannose content is about 5-25% by mass. According to the study by the present inventors, the effect of inducing the expression or promoting the production of antibacterial peptides in gastrointestinal tissues is not obtained simply by using a mannan-containing substance. It has been found that when a mannan-containing component derived from yeast, which is 60% by mass or more, is used, it can be obtained specifically and strongly. From such a viewpoint, the mannose content of the mannan-containing component derived from yeast is preferably 60% by mass or more, and more preferably 70% by mass or more.

  It is known that the structure and components of mannan vary depending on the origin, and in the present invention, the mannan-containing component derived from yeast has a mannose content of 60% by mass or more. This is based on the finding that the induction or production promoting effect is high.

  As described above, the promoter of the present invention uses the physiological function of the mannan-containing component derived from yeast. As the starting material for the promoter of the present invention, any starting material can be used as long as it contains a yeast cell wall. For example, it can be obtained by using yeast cells or a culture solution as a starting material. . More specifically, either yeast cells, yeast cell extracts that are enriched with mannan compared to cells, or yeast culture supernatants containing mannan released from or secreted from yeast cells. May be used. Whether the yeast cells are live or dead, it is preferable to use a mannan content of 5% (w / w) or more as a raw material in the analysis in a dry state. This is desirable from the viewpoint of obtaining a mannan-containing component having a high mannose content. As yeast, for example, Saccharomyces beer yeast and baker's yeast are known. As for the yeast cell extract and culture supernatant, it is desirable to use a mannan content of 25% (w / w) or more as a raw material in the analysis in a dry state.

Description of the extract When preparing a cell extract used as a raw material of the promoter of the present invention, it is preferable to use a yeast cell of the genus Saccharomyces as a raw material, which contains live or dead bacteria or water. It does not matter whether it is dry or not. Further, mannan may be further extracted from a yeast extract prepared other than for the purpose of concentrating mannan, or a yeast cell wall obtained as an insoluble residue when the yeast extract is produced. In this case, you may use a commercial item for a yeast extract or a cell wall. Examples of yeasts belonging to the genus Saccharomyces include baker's yeast and beer yeast, but the yeast to be used is not limited to these.

  In order to prepare the cell extract by extracting or concentrating mannan from cells of Saccharomyces yeast, for example, water or an organic solvent is used, and a mixture of these may be used. A preferred extraction solvent is acidic or alkaline water. Extraction is performed by room temperature extraction, heating extraction, or pressure extraction (processing such as autoclave). Generally, it is performed at room temperature to 121 ° C. After the extraction process, the solid content and the liquid are separated by centrifugation or the like to obtain only the liquid part. In order to remove unnecessary low-molecular components such as salts from this solution, a low-molecular fraction may be removed by membrane treatment, or ethanol or the like may be added to precipitate soluble carbohydrates and recovered. The obtained mannan-containing extract solution can be concentrated by vacuum concentration or the like, if necessary, or powdered by vacuum drying, freeze drying, or the like. An appropriate excipient may be added during powdering. In addition, in the case of precipitation by addition of ethanol or the like, the precipitate may be washed with hydrous ethanol or the like and dried or powdered as it is, or ethanol or the like may be removed to form a solution. After extraction by the above method, the mannan-containing extract obtained by membrane treatment or ethanol precipitation has a mannose or polymer content of 60% by mass or more.

  The method of hot water treatment for extracting water-soluble polysaccharides from yeast cells can be, for example, a method of hot water extraction of yeast cells and yeast self-digesting insoluble matter at high temperature. Examples of the extraction solvent include water or phosphate buffer (pH 6.0 to 8.0). Extraction can be performed under normal pressure, or preferably under a pressure of 2 to 5 atmospheres. Although the heating temperature and the time vary depending on the extraction method, it is appropriate to perform the heating at 90 to 100 ° C. for 10 to 20 hours as a suitable condition. As a method for separating mannan from the extract, for example, a method of solid-liquid separation of the extract and ultrafiltration of the resulting supernatant can be employed. As the ultrafiltration membrane, it is preferable to use a filtration membrane having a molecular weight fraction of about 5000 to 30000. Next, after removing a small amount of cold coagulum generated by leaving the impervious liquid that has not passed through the filtration membrane at a low temperature (for example, the temperature in the refrigerator) for 24 hours, the impervious liquid is dried to obtain white yeast. A powder containing mannan-containing extract is obtained. Alternatively, after the solid-liquid separation, the resulting supernatant is filtered, the filtrate is concentrated under reduced pressure, left to stand at low temperature, the cold coagulum is removed by filtration, and the filtrate is added to ethanol to obtain By drying the resulting precipitate, a yeast mannan-containing extract powder can be obtained.

  The thus obtained yeast-derived mannan-containing material (powder) (accelerator of the present invention) has a content of mannose or a polymer thereof of 60% by mass or more. Furthermore, the mannan-containing material (powder) derived from yeast is superior in water solubility compared to mannan derived from konjac, and the viscosity of the aqueous solution of mannan-containing material (powder) derived from yeast is lower than mannan derived from konjac. . That is, the mannan-containing material (powder) derived from yeast has water solubility enough to dissolve at least 15 g in 100 mL of distilled water at room temperature, for example. The viscosity of the aqueous solution of mannan-containing material (powder) derived from yeast is 6.0 cps or less with a tuning fork viscometer when dissolved in distilled water at 25 ° C. to give a 10% solution.

  The accelerator of the present invention can also be in a liquid form, and in that case, 100 mg to 2 g of mannose can be contained per 100 mL.

  The promoter of the present invention is used as an antibacterial peptide to induce expression or promote production of α-defensin or β-defensin, and thus is expected to have an immunostimulatory effect, an infectious disease preventive effect, and / or an AIDS onset suppressive effect. The In any case, administration of the promoter of the present invention promotes the expression induction or production of α-defensin or β-defensin in the living body, particularly in the intestinal tract. As a result, immunostimulatory action, infection prevention action and / or AIDS onset suppression effect is obtained. Although the detailed mechanism of the immunity improving action is not clear, the immunostimulatory action in the present invention is not due to acquired immunity (or adaptive immunity) involving antibodies or the like as described above, but early immunity involving defensins. It is thought to be due to.

The present invention relates to a pharmaceutical or a food or drink containing the promoter of the present invention.
The promoter of the present invention can be used as a pharmaceutical product as an oral product, and can be mixed with a food material to make a food or drink. The properties are solid or liquid, and pharmaceuticals can be in the form of tablets, capsules, granules, syrups and the like as oral preparations.

  When administered to the human body as a pharmaceutical, the amount of 1 to 500 mg (dry weight) / kg body weight per dose, preferably 2 to 100 mg (dry weight) / kg body weight is orally administered once to several times a day can do.

  In the case of a food or drink, the food material to be mixed may be a solid (powder, flake, lump, etc.), a semi-solid (jelly, syrup, etc.), or a liquid. As for content per 1g of food-drinks, it is desirable that it is 1-500 mg (dry weight). The example of the food / beverage in the case of setting it as food / beverage is not specifically limited.

  Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited thereto.

Example 1 (Preparation of yeast mannan from Saccharomyces yeast)
1000 g of beer yeast cell walls (Asahi Food & Healthcare) were suspended in distilled water, adjusted to pH 11.0 by adding sodium hydroxide, and then boiled at 90 ° C. for 12 hours. The supernatant was recovered by centrifugation and adjusted to pH 4.0 by adding hydrochloric acid. After high-pressure sterilization, heat coagulated substances and the like were removed, and sodium hydroxide was added to the obtained supernatant to adjust the pH to 5 or more. By freeze-drying, 15 g of yeast mannan fraction was obtained. As a result of analysis by sulfuric acid hydrolysis / high performance liquid chromatography, the ratio of mannose in the yeast mannan fraction was 77% (w / w).

Example 2 (Production of yeast mannan-containing extract)
0.05 g phosphorus is added to 5.0 g of dried yeast self-digestible insoluble matter obtained by spray-drying the residue obtained by removing the water-soluble yeast extract produced by self-digestion of brewer's yeast (Saccharomyces cerevisiae) 100 ml of an acid buffer (pH 7.5) was added, and the mixture was extracted by heating at 100 ° C. for 16 hours. After cooling, solid-liquid separation was performed by centrifugation, and the supernatant was ultrafiltered (ULTRA FILTER molecular fraction 20000, manufactured by ADVANTEC). The impervious liquid that did not pass through the filtration membrane was cooled to 4 ° C., and the coagulum was removed by centrifugation. The supernatant was lyophilized to obtain a yeast mannan-containing extract. The yeast mannan-containing extract had a mannose content of 84% w / w (dry weight).

(Water-soluble yeast mannan-containing extract)
The obtained mannan-containing extract derived from yeast was highly water-soluble and could be dissolved in an amount of 15 g or more in 100 ml of distilled water at room temperature.

(Viscosity of yeast mannan-containing extract)
When the obtained mannan-containing extract (powder) derived from yeast was dissolved in distilled water at 25 ° C. to give a 10% solution, the viscosity was 6.0 cps or less with a tuning fork viscometer. Furthermore, even when it was 20% solution, it did not become a gel and maintained high water solubility.

Comparative Example 1
(Water-soluble konjac mannan)
Konjac mannan was less than 1 g dissolved in 100 ml of distilled water at room temperature.
(Viscosity of konjac mannan)
The viscosity when konjac mannan was suspended in distilled water at 25 ° C. to form a 1% solution was 103 cps or more with a tuning fork viscometer.

Example 3 (Infectious protective effect of yeast mannan)
Six-week-old BALB / c male mice (Japan SLC) were divided into two test groups consisting of five mice per group, and the standard feed CRF-1 (control group) or the yeast mannan fraction obtained in Example 1 was 0.5. % Feed (mannan group) was ingested freely. One month later, dissection was performed, and in addition to the liver and spleen, gastrointestinal tissues (mesenteric lymph nodes, Peyer's patches, jejunum, ileum, colon, rectum, cecum) were collected. The collected tissue was pulverized using a homogenizer or the like, and then total RNA was extracted using RNeasy mini kit (QIAGEN). CRNA labeled with Cyanine 3-CTP or Cyanine 5-CTP was prepared from total RNA using Low RNA Input Fluorescent Linear Amplification Kit (Agilent). The prepared cRNA was used in a Mouse Oligo Microarray Kit (Agilent), and a comprehensive gene expression investigation was performed by the DNA microarray method. Changes in the expression level of each gene in the mannan group relative to the control group were evaluated by the dye exchange method.

  When the expression of defensin-related genes was investigated in each tissue, it was found that the expression of defensin-related genes in the yeast mannan intake group increased 1.5 times or more when the control group that received standard feed was 1. 1 was found.

Example 4 (tablets, capsules)
Yeast mannan fraction (Example 1) 10.0 g
Lactose 75.0g
Magnesium stearate 15.0g
Total 100.0g
The above respective parts by weight were uniformly mixed, and tablets and capsules were prepared according to a conventional method.

Example 5 (powder, granule)
Yeast mannan fraction (Example 1) 20.0 g
Starch 30.0g
Lactose 50.0g
Total 100.0g
Each said weight part was mixed uniformly, and it was set as the powder and the granule according to the conventional method.

Example 6 (飴)
Sucrose 10.0g
Chickenpox (75% solids) 60.0g
9.5g of water
Coloring 0.455g
Perfume 0.045g
Yeast mannan fraction (Example 1) 20 g
Total 100.0g
Using each component of each of the above parts by weight, it was made into a candy according to a conventional method.

Example 7 (juice)
150g concentrated orange juice
Fructose 50g
Citric acid 2g
1g fragrance
Color element 1.5g
Sodium ascorbate 0.5g
Yeast mannan fraction (Example 1) 10 g
785 g of water
Total 1000g
Using each component of the above-mentioned parts by weight, juice was prepared according to a conventional method.

Example 8 (barley tea)
Barley tea 5.0g
2995 g of water
Total 3000g
Using each component of each of the above parts by weight, after extracting barley tea components according to a conventional method,
Sodium ascorbate 1.8g
Yeast mannan fraction (Example 1) 40 g
To make barley tea.

Example 9 (cookies)
Soft flour 32.0g
Whole egg 16.0g
16.0g butter
Sand sugar 25.0g
10.8g of water
Baking powder 0.198g
Yeast mannan fraction (Example 1) 0.002 g
Total 100.0g
Using each component of the above-mentioned parts by weight, a cookie was prepared according to a conventional method.

  Since the pharmaceuticals and foods and drinks containing the mannan-containing component of the yeast of the present invention have an action of promoting expression induction or production of antibacterial peptides in the gastrointestinal tract tissue, they have an immunostimulatory action, an infectious disease preventive action, or an AIDS crisis-suppressing action. It is effective in the prevention and treatment of diseases and is useful in the fields of medicine and food.

Claims (9)

An agent for inducing or promoting the expression of antibacterial peptides in gastrointestinal tissues, comprising a mannan-containing component derived from yeast as an active ingredient. The accelerator according to claim 1, wherein the mannan-containing component has a mannose content of 60% by mass or more. The promoter according to claim 1 or 2, wherein the mannan-containing component is obtained from a yeast cell or a culture solution. The promoter according to any one of claims 1 to 3, wherein the yeast is a brewery yeast. The accelerator according to any one of claims 1 to 4, wherein the accelerator is water-soluble, and the aqueous solution of the accelerator has a low viscosity. The accelerator according to claim 5, which is liquid and contains 100 mg to 2 g of mannose per 100 mL. The promoter according to claim 1, which is used for inducing expression or promoting production of α-defensin or β-defensin as the antimicrobial peptide. The promoter according to any one of claims 1 to 7, which has an immunostimulatory action, an infectious disease preventive action, and / or an AIDS onset suppressing action. The pharmaceutical or food-drinks containing the promoter in any one of Claims 1-8.