JP2005082494A - Mucous membrane-immunostimulating composition - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a safe and inexpensive mucous membrane-immunostimulating composition, and to provide a mucous membrane-immunostimulating food, drink and medicine each containing the composition. <P>SOLUTION: This mucous membrane-immunostimulating composition containing a phosphorylated saccharide is characterized in that the saccharide is a glucan and is bound to a plurality of phosphate groups per one glucan molecule. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a mucosal immunostimulatory composition comprising phosphorylated saccharide.

  Skin and mucous membranes, which are the borders between which the living body comes into contact with the outside world, in particular, gastrointestinal mucosa such as intestinal mucosa, come into contact with many substances such as viruses, bacteria, parasites, pathogenic antigens, and food antigens. In the gastrointestinal tract immune system for protecting living organisms, immunoglobulin A (IgA) is known as one having the action of preventing the entry of foreign substances. IgA neutralizes bacteria and viruses, and acts on tissues. It plays an important role in the suppression of bacterial adhesion and the suppression of allergies caused by food antigens. It is known that the production of IgA involves Peyer's patch lymphocytes, which are lymphoid tissues in the intestinal tract (see Non-Patent Document 1).

  When the immune system of the defense system in the gastrointestinal mucosa is reduced, symptoms such as fever, vomiting, diarrhea, and abdominal pain occur due to gastrointestinal infection caused by colonization and proliferation of microorganisms in the gastrointestinal tract. In addition, anorexia due to overwork, stress, etc. also tends to reduce gastrointestinal immunity and cause bacterial diarrhea and the like (see Non-Patent Document 2).

  In particular, infants, elderly people, people with weak physical strength, etc. have weak immunity and have low resistance to invasion of pathogenic bacteria from the outside. Therefore, there is provided a composition having an action capable of enhancing the production of IgA, which plays an important role in neutralizing bacteria and viruses, inhibiting bacterial adhesion to tissues, and inhibiting allergies caused by food antigens, and keeping the amount high. It is desired.

  As such a composition for enhancing IgA production, for example, casein phosphopeptide (hereinafter abbreviated as CPP) and a composition thereof have been proposed. CPP is known to have the action of maintaining the solubilized state of calcium insolubilized in the small intestine and promoting calcium absorption, and has already been put into practical use as a raw material for many foods or feeds. In addition, CPP is known to have an effect of enhancing IgA production in cultured cell lines or in the digestive tract of animals (see Non-Patent Document 3 and Non-Patent Document 4), and further added with CPP. It has been shown that IgA levels in feces are also enhanced when humans take tablet confections (see Non-Patent Document 5).

  However, since CPP is expensive, its usage is limited and its usage is limited. Moreover, since CPP is obtained by degrading casein with an enzyme, its antigenicity is lower than that of casein, but it has not completely disappeared. For this reason, if a person who is allergic to milk casein expects to enhance immunity and ingests CPP, there is concern that an allergic reaction will be caused.

  In addition, no prophylactic and therapeutic agents that have an IgA production-enhancing action and are effective against reductions in gastrointestinal immunity function, bacterial gastrointestinal tract infection, etc. are currently known, such as actual bacterial diarrhea In contrast, only infusion and antibiotic administration for dehydration are performed (see Non-Patent Document 6).

Hajime Otani, Food and Technology, 135, 1-9 (2003) General Protecting yourself from esophageal and gastrointestinal diseases Part 2; Life Daily, March 2002 Hajime Otani; Milk Science, 49, 73-79 (2000) Hajime Otani ； Food and Agricultural Immunology, 12, 165-173 (2000) Nakano et al .: Abstracts of the 99th Annual Meeting of the Japanese Society of Animal Science (2001) Special issue: Infectious diarrhea ； Pharmaceutical Journal, 35 (5), 67-104 (2000)

  An object of the present invention is to provide a safe and inexpensive mucosal immunostimulatory composition, and a mucosal immunostimulatory food / beverage product and pharmaceutical comprising the composition.

  As a result of intensive studies in view of the above circumstances, the present inventors have found that when orally ingesting phosphorylated glucan, (1) the total amount of IgA in stool increases significantly, (2) antigen-specific in stool The inventors have found that the amount of IgA increases, (3) the production amount of IgA in the intestine increases, and (4) the amount of antigen-specific IgA production in the intestine increases, thereby completing the present invention. These results indicate that phosphorylated glucan stimulates immunity by inducing IgA production in the intestinal lumen.

That is, the present invention includes the following inventions.
(1) A mucosal immunostimulatory composition comprising phosphorylated saccharide, wherein the saccharide is a glucan and a plurality of phosphate groups are bound per molecule of glucan.
(2) The mucosal immunostimulatory composition according to (1) for enhancing IgA production ability.
(3) The mucosal immunostimulatory composition according to (1) or (2), which protects against infection caused by infectious diseases.
(4) Mucosal immunostimulatory food or drink containing the mucosal immunostimulatory composition according to any one of (1) to (3).
(5) Mucosal immune-stimulated food or drink according to (4), which is infant formula, infant food, health functional food, nutritional supplement, or sick food.
(6) A mucosal immunostimulatory pharmaceutical comprising the mucosal immunostimulatory composition according to any one of (1) to (3).
(7) The mucosal immunostimulatory drug according to (6), further comprising a pharmaceutically acceptable additive.

  According to the present invention, a safe and inexpensive mucosal immunostimulatory composition, and a mucosal immunostimulatory food / beverage product and pharmaceutical comprising the composition are provided.

The mucosal immunostimulatory composition according to the present invention includes phosphorylated glucan.
Since the starting material of phosphorylated glucan, which is an active ingredient of the mucosal immunostimulatory composition according to the present invention, is sugar, it goes without saying that its safety is generally low and easy to obtain. Is also advantageous. In Japan, phosphoric acid ester starch is approved as a food additive as phosphorylated starch and is widely used in food.

  In the present invention, “glucan” has a normal meaning in the art, that is, a polycondensation polymer of D-glucopyranose. In the present invention, the glucan is usually composed of 3 to 300, preferably 6 to 250, more preferably 9 to 100 glucose residues. In the present invention, the glucan may be either α-glucan or β-glucan, but α-glucan is preferred. In addition, glycosidic bonds in glucan include α-1,3 bonds, α-1,4 bonds and α-1,6 bonds, and β-1,3 bonds, β-1,4 bonds and β-1,6 bonds. Any of these may be included. The glucan may be branched or unbranched.

  In the present invention, naturally occurring glucan is preferable. The glucan in the present invention includes, for example, cellulose, amylose, amylopectin, glycogen, starch, dextrin and the like, and processed products obtained by decomposing these by chemical or enzymatic treatment. In the present invention, dextrin is preferred. Examples of dextrin include amylodextrin, erythrodextrin, acrodextrin, maltodextrin, cellodextrin, cyclodextrin and the like, and maltodextrin is particularly preferable.

  The phrase “a plurality of phosphate groups are bonded per molecule of glucan” means that two or more phosphate groups are bonded per molecule of glucan composed of a plurality of glucose units. The number of phosphoric acid units to be bound varies depending on the number of glucose residues constituting one molecule of glucan, but when converted to 100 glucose residues, 2 to 40, more preferably 10 to 30 phosphoric acids. It is preferred that the groups are bonded. The phosphate group can be ester-bonded at any position other than the glycosidic bond position in the glucopyranose ring of glucan.

  The use of the mucosal immunostimulatory composition of the present invention is not limited to foods and drinks, and can be blended and used in pharmaceuticals.

  In the present invention, the “mucosal immunostimulatory composition” means a composition that has mucosal immunostimulatory activity, ie, activity that enhances mucosal immunity and can be used to enhance mucosal immunity. “Mucosal immunostimulatory activity” means an activity that enhances immunity in the mucous membrane of the digestive organ, respiratory organ or body surface. The mucosal immunostimulatory composition of the present invention has immunostimulatory activity in the digestive tract, particularly in the intestinal mucosa.

  The mucosal immunostimulatory composition of the present invention has an activity of increasing IgA in feces and intestinal tract particularly in animals including humans. Since the immunoglobulin A (IgA) contained in the mucosal secretion plays an important role as one of the local immune mechanisms in the mucosa, the mucosal immunostimulatory composition of the present invention has an excellent mucosal immunostimulatory activity. Have Therefore, the mucosal immunostimulatory activity of the composition of the present invention can be evaluated by the activity of increasing IgA in stool or intestinal tract in animals including humans. Here, IgA includes both total IgA and antigen-specific IgA, and the mucosal immunostimulatory composition of the present invention has an activity of increasing both. For example, when the composition is administered to an animal under the same conditions as in the examples of the present specification and total IgA and antigen-specific IgA in feces and intestinal tract are measured, The product can be evaluated as having mucosal immunostimulatory activity.

  The phosphorylated glucan according to the present invention can be produced by using various conventional synthetic methods using a sugar of natural origin or non-natural origin as a starting material. For example, although it can manufacture according to the following procedures, it is not limited to these.

  Particulate glucan from a microbial source (eg, Saccharomyces cerevisiae) is suspended in a highly polar solvent containing a strong chaotropic agent, and the resulting mixture is reacted with phosphoric acid with constant stirring at about 50-150 ° C. After about one hour, a reaction mixture containing a soluble phosphorylated glucan is produced.

  In order to increase the yield of active ingredient, it is preferred to hold the reaction mixture at about 100 ° C. for about 3-12 hours. Soluble phosphorylated glucan can be isolated by performing filtration to remove residual sediment from the reaction mixture, molecular sieving to remove a substance having a molecular weight smaller than the target substance, dialysis, and the like. Thereafter, a soluble phosphorylated glucan is finally obtained by concentration and freeze-drying (see Japanese Patent No. 2550332).

  Moreover, the following method is mentioned as a preferable method for obtaining the phosphorylated saccharide | sugar of this invention. That is, a saccharide originating from a natural product or a non-natural product is prepared, and the saccharide is heated in a phosphate buffer, for example, at 50 to 120 ° C. for 5 to 30 minutes, preferably at about 80 ° C. for 10 minutes. To react. At this time, the sugar concentration is usually 0.5 to 10%, preferably 1 to 5%. After the heat treatment, the treatment mixture is freeze-dried, and the resulting freeze-dried product is further heat-treated at 90 to 160 ° C., preferably about 110 to 140 ° C. for 12 to 36 hours, preferably 24 hours. Then, if desired, the heat-treated product is purified by dialysis or the like to obtain the target phosphorylated saccharide.

  Furthermore, known methods for producing dextrin phosphate can also be used. For example, as disclosed in JP-A-11-255803, phosphate oligosaccharides (within 10 glucose residues) obtained by allowing α-amylase to act on phosphate starch, dextrin phosphate (10 glucose residues) Ultra) can also be a method for producing a target phosphorylated saccharide.

  The present invention also relates to a mucosal immunostimulatory food or drink containing the mucosal immunostimulatory composition. The mucosal immunostimulatory food or drink of the present invention is not particularly limited, and can be provided in the form of gum, biscuits, chocolate, candy, jelly, tablet confectionery, powdered milk, beverages, and the like.

  The mucosal immunostimulatory food / drink of the present invention can be used for infant formulas and follow-up milk for infants with immature immune function development, foods for the elderly with decreased immune function, patients after illness, and allergic diseases. It can be produced by adding an effective amount of a mucosal immunostimulatory composition as a food and drink for nutritional supplementation and health promotion of patients with various diseases. It can also be applied to health functional foods and foods for the sick. Functional health foods are intended not only for regular foods, but also for foods in the form of tablets, capsules, etc., based on domestic and external trends and consistency with the conventional food system for specified health use. It consists of two types of foods for health (individual permission type) and nutritional functional foods (standard standard type).

  The content of the mucosal immunostimulatory composition in the mucosal immunostimulatory food or drink can be determined as appropriate in consideration of the purpose of use. For example, the dose to humans is usually 1 to 1000 mg / kg body weight, preferably 10 to 400 mg / kg body weight per day, as phosphorylated glucan as an active ingredient.

  The present invention also relates to a mucosal immunostimulatory pharmaceutical comprising the mucosal immunostimulatory composition of the present invention. The mucosal immunostimulatory pharmaceutical of the present invention can be made into a suitable dosage form according to the administration route. Specifically, injections such as intravenous injection and intramuscular injection, capsules, tablets, granules, powders, pills, fine granules, dragees, troches, chewable tablets, etc., oily suppositories, etc. The dosage form can be prepared. In addition, the mucosal immunostimulatory pharmaceutical of the present invention may be in the form of a liquid, a suspension, a capsule containing a liquid, etc., if necessary.

  The mucosal immunostimulatory pharmaceutical according to the present invention may further contain a pharmaceutically acceptable additive. Examples of pharmaceutically acceptable additives include excipients, fillers, binders, wetting agents, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, solubilizers, Preservatives, flavoring agents, soothing agents, stabilizers and the like can be mentioned. The mucosal immunostimulatory pharmaceutical of the present invention can be produced by a conventional method by blending these additives as necessary.

  Specific examples of the additive include, for example, lactose, fructose, glucose, gelatin, magnesium carbonate, synthetic magnesium silicate, talc, magnesium stearate, methylcellulose or a salt thereof, gum arabic, polyethylene glycol, citric acid, sodium chloride, Examples include sodium sulfite and sodium phosphate.

  The mucosal immunostimulatory pharmaceutical of the present invention can be administered in various administration forms such as oral administration, parenteral administration, inhalation, enteral administration, and local administration. Among these, parenteral administration includes subcutaneous injection, intravenous administration, intramuscular administration, intranasal administration or infusion. Therefore, for example, mucosal administration such as nasal, oral, sublingual, rectal, etc., transdermal administration, or administration by an implant may be used, and the mucosal immunostimulatory drug according to the present invention is administered by any of these administration routes. Can do.

Diseases for which prevention, treatment or improvement effects are expected by the mucosal immunostimulatory drug of the present invention include pancreatic cancer, stomach cancer, colon cancer, rectal cancer, lung cancer, endometrial cancer, cervical cancer, lymphoma, leukemia and the like. Examples include cancer, gastric ulcer, hepatitis, collagen disease, rheumatoid arthritis, psoriasis, hay fever, immune diseases such as atopic dermatitis, bacterial or viral infections, and the like. Among these, it is suitably used for the prevention, treatment or improvement of infections caused by bacteria or viruses, particularly infections caused by Salmonella bacteria. Salmonella bacteria include S. choleraesuis, S. typhi,
S. Oranienburg, S. chester, S. Enteritidis, S. Choleraesuis and so on.

  The dose of the mucosal immunostimulatory drug may be appropriately determined in consideration of the usage, patient age, sex, symptom level, etc. The determined amount may be administered once or several times a day. it can. For example, the dose to humans is usually 1 to 1000 mg / kg body weight, preferably 10 to 400 mg / kg body weight per day, as phosphorylated glucan as an active ingredient.

  EXAMPLES Hereinafter, although an Example demonstrates in detail about this invention, this invention is not limited by this.

[Example 1] Preparation of phosphorylated dextrin Dextrin (molecular weight 2700, Sanwa Starch Kogyo Co., Ltd.) was adjusted to 80 ° C so as to have a concentration of 2% in 0.1M phosphate buffer (pH 5.5). And dissolved in a water bath with shaking for 10 minutes. This solution was freeze-dried, transferred to an Erlenmeyer flask, and kept for 24 hours in a dryer adjusted to 140 ° C. without a lid. Subsequently, it melt | dissolved in the demineralized water so that it might become a 2% density | concentration, it inject | poured into the PPECTRA / PORT tube (cut off MW500, the product made from Funakoshi Co., Ltd.), and dialyzed against tap water for 72 hours. Next, the lyophilized dextrin was used as phosphorylated dextrin.

  The amount of organic phosphoric acid contained in one molecule of phosphorylated dextrin was determined by measuring the amount of organic phosphorous contained in a certain amount of phosphorylated dextrin. First, in order to determine the amount of inorganic phosphoric acid, weighed phosphorylated dextrin into a 25 ml volumetric flask, 2.0 ml of 70% perchloric acid, 2.0 ml of amidol (2,4-diaminophenol hydrochloride) reagent and 8 After adding 1.0 ml of 3% ammonium molybdate solution and mixing well, it was made up to 25 ml with demineralized water. Next, after standing at room temperature for 20 minutes, the absorbance at a wavelength of 720 nm was measured using UV-VIS SPECTROTOPOMETER (manufactured by Shimadzu Corporation). A standard solution containing a known amount of dipotassium hydrogen phosphate was measured by the same method, and the amount of inorganic phosphate was calculated by substituting the absorbance into the prepared standard curve.

  On the other hand, in a 30 ml Kjeldahl decomposition flask, 2.2 ml of a fixed amount of phosphorylated dextrin dissolved in water and 2.2 ml of 70% perchloric acid were added, and the solution was mixed for 15 to 25 minutes using a Kjeldahl decomposition apparatus. Heat until the color is constant. After allowing to cool once, 20 μl of 30% aqueous hydrogen peroxide was added and heated again for 5 minutes. Next, 3.0 ml of demineralized water was added to the decomposed liquid which had been allowed to cool, and it was immersed in a boiling water bath for 10 minutes for complete wet decomposition. Subsequently, this solution was transferred to a 25 ml volumetric flask, and after adding 2.0 ml of Amidol reagent and 1 ml of 8.3% ammonium molybdate solution, the absorbance was measured in the same manner as inorganic phosphoric acid, and the obtained absorbance was standardized. The value calculated by assigning to the curve was defined as the total phosphoric acid amount. The organic phosphoric acid amount was determined by subtracting the inorganic phosphoric acid amount from the total phosphoric acid amount.

  According to the above method, 4 to 5 phosphoric acids were detected per molecule of phosphorylated dextrin. That is, the phosphorylated glucan obtained in this example was composed of an average of 15 glucoses and contained 4 to 5 phosphoric acids per molecule.

[Example 2]
Using the phosphorylated dextrin prepared in Example 1, the effect on IgA antibody production-induced mice was examined.
(1) Materials and Methods The experimental animals used were male C3H / HeN mice purchased (SLC) at 5 weeks of age. Phosphorylated dextrin was prepared according to Example 1. Protein adjusted feed (PM15765) was purchased from Purina Mills. Salmonella LPS was used after being dissolved in sterilized phosphate buffered saline (hereinafter sometimes abbreviated as PBS). The animals were divided into two groups (5 per group): a control group and a phosphorylated dextrin group. From the date of purchase to the end of the test, the control group was allowed to freely ingest the basic diet, and the phosphorylated glucan group was allowed to freely consume the diet in which dextrin was replaced with an equal amount of phosphorylated dextrin (Table 1). Drinking water was tap water ad libitum. Moreover, Salmonella LPS (lipopolysaccharide) was forcibly orally administered using a sonde at 7, 14 and 21 days after the start of breeding, 10 μg per individual. The body weight of the mice was measured 0, 14, 21, 28, and 35 days after the start of breeding.

(2) Preparation of IgA measurement sample in stool The whole amount of stool was collected from the cage 0, 14, 21, 28, and 35 days after the start of the test. To 2 g of the collected feces, 3 g of sea sand and 5 ml of sterilized PBS were added, both were ground in a mortar for 15 minutes, and centrifuged at 4 ° C. and 3000 rpm for 20 minutes. The obtained supernatant was collected and used as a fecal extract.

(3) Preparation of IgA measurement sample in intestinal tract tissue On the 35th day from the start of the test, the mouse was dissected and the duodenum to the rectum was removed. 3 g of sea sand and 5 ml of sterilized PBS were added to 2 g of the collected intestinal tract, both were ground for 15 minutes in a mortar, and centrifuged at 4 ° C. and 3000 rpm for 20 minutes. The obtained supernatant was recovered and used as an intestinal extract.

(4) Measurement of total IgA and Salmonella LPS-specific IgA IgA was measured by a sandwich ELISA method (Williams, DJLet al., Vet. Immunol. Immunopath., 24, 267-283 (1990)). Total IgA was determined by adding an appropriately diluted measurement sample to a plate coated with goat anti-mouse IgA antibody, washing, and reacting with anti-mouse IgA antibody labeled with horseradish peroxidase. LPS-specific IgA was determined by adding an appropriately diluted measurement sample to a plate coated with Salmonella LPS, washing, and reacting with anti-mouse IgA antibody labeled with horseradish peroxidase. Detection was carried out by measuring the absorbance at 490 nm, and the product multiplied by the dilution factor of the sample solution was used as the ELISA value. Student's t-test was used to determine the significant difference in effect. The total IgA and Salmonella LPS-specific IgA contents in feces are shown in Table 2, and the total IgA and Salmonella LPS-specific IgA contents in intestinal tissue are shown in Table 3, respectively.

(5) Results and Discussion With the ingestion of phosphorylated dextrin, the total amount of IgA in the stool increased significantly from 14 days after the start of the breeding, and the LPS-specific IgA level from 21 days later (Table 2). In addition, ingestion of phosphorylated dextrin significantly increased the total amount of IgA in the intestinal tissue and the amount of LPS-specific IgA (Table 3). There was no significant difference in weight gain during the test period between the control group and the phosphorylated dextrin group. From the above, it was revealed that phosphorylated dextrin induces IgA production in the intestine and has mucosal immunostimulatory activity.

  By adding the mucosal immunostimulatory composition of the present invention to infant formula, foods for the elderly, health functional foods (specific health foods and nutritional functional foods), nutritional supplements, foods for the sick, or pharmaceuticals Mucosal immunostimulatory foods and drinks and mucosal immunostimulatory pharmaceuticals can be provided.

Claims (7)

  A mucosal immunostimulatory composition comprising a phosphorylated saccharide, wherein the saccharide is a glucan and a plurality of phosphate groups are bound per molecule of glucan.   The mucosal immunostimulatory composition according to claim 1 for enhancing IgA production ability.   The mucosal immunity activation composition of Claim 1 or 2 for protecting the infection by an infectious disease.   Mucosal immunostimulatory food or drink containing the mucosal immunostimulatory composition according to any one of claims 1 to 3.   The mucosal immune-stimulated food or drink according to claim 4, which is a formula for infants, a food for elderly people, a health food, a dietary supplement, or a food for the sick.   A mucosal immunostimulatory pharmaceutical comprising the mucosal immunostimulatory composition according to any one of claims 1 to 3. The mucosal immunostimulatory drug according to claim 6, further comprising a pharmaceutically acceptable additive.