JP4782385B2 - Immunostimulator - Google Patents

Description

  The present invention relates to an immunostimulant containing saccharide as an active ingredient. More specifically, the present invention relates to macrophage interleukin 12 (hereinafter referred to as “active ingredient”) containing saccharides that have not been known to have an immunostimulatory effect, such as D-mannose, isomaltose, laminaribiose, and cellobiose as active ingredients. The present invention relates to an immunostimulant having an IL-12) producing ability enhancing action or an interferon γ (hereinafter, IFN-γ) producing ability enhancing action in intestinal interepithelial cell lymphocytes.

  Living organisms such as humans are constantly exposed to invading foreign microorganisms and foreign self-substances such as endogenous tumor cells. An immune system is provided as a biological defense mechanism for combating various diseases caused by these diseases. The types of immune responses can be broadly classified into cellular immunity, in which immune cells themselves attack and eliminate foreign substances, and humoral immunity, in which foreign substances are eliminated by antibodies produced by immune cells.

  One immune system cell that plays an important role in the early stages of the immune response is macrophages. Macrophages selectively prey on invading microorganisms and unnecessary apoptotic cells and necrotic cells generated in the morphogenesis process of an individual or in the body of the living body, digest them, and present these as antigens to lymphocytes. In addition, there are receptors on the surface of macrophages that transmit stimuli by pattern recognition of sugar chains called Toll-like receptors. In these processes, macrophages produce various cytokines and chemokines and control other cellular functions involved in biological defense responses. For example, interleukin 12 (IL-12) produced by macrophages activates natural killer (NK) cells and also differentiates naive T cells that have not been antigen-sensitized into Th1, thereby making Th1 / Th2 balance superior to Th1. It is known that it has the effect of leading the immunity mode to cellular immunity. Furthermore, IFN-γ produced by Th1 cells is known to have an action of activating NK cells and killer T cells, an action of inducing macrophage bactericidal activity, and the like. Therefore, IL-12 produced by macrophages is triggered, and immune cells of the cellular immune system are activated through production of IFN-γ by Th1 cells.

  In recent years, in Japan, an aging society is accelerating, and there are many cases in which elderly people die due to viral or microbial infections due to a decrease in immunity due to aging. In addition, it is known that irregular intake of meals in a busy modern society and an environment susceptible to stress may cause a decrease in immunity, and countermeasures are strongly desired. As a countermeasure, new antitumor agents, vaccines, and antibiotics have been actively developed. However, these therapeutic agents are problematic in that they have side effects, and recently, medical expenses have increased rapidly, and there is a strong demand for reducing medical expenses by improving meals.

  Since saccharides have a long dietary experience, they can be a relatively safe therapeutic agent if they have some physiological activity. For example, D-mannose is recognized in vivo as a constituent component of a sugar chain as seen in high-mannose sugar chains, and plays an important role in maintaining life activity. It is being done. In addition, D-mannose is known to have an action of inhibiting Salmonella colonization in chicken intestinal epithelial cells (Non-patent Document 1), and is used as a feed additive for preventing infection of harmful bacteria in the field of livestock. Has been proposed (Patent Documents 1 and 2).

However, IL-12 and IFN, which are cytokines that activate immune cells (macrophages, killer T cells, NK cells, etc.) of the cellular immune system that play a role in protecting against pathogenic microorganisms and viruses, and destroying tumor cells It has not been conventionally known that production of -γ is enhanced by these carbohydrates. So far, sugar chains having such functions include nigerooligosaccharide (Non-patent Documents 2 and 3), raffinose (Patent Documents 4 and 5), neoagaro-oligosaccharide (Patent Document 6), and β1,3. Only glucan (Patent Document 7) has been reported. In addition, about the mixture of isomaltooligosaccharide, although the effect | action which increases T lymphocyte and B lymphocyte is confirmed, it is not known at all about the production enhancement of IL-12 and IFN-gamma (patent document 8). .
JP 2001-213781 A JP-A-8-38064 JP 2002-80364 A JP 2001-288093 A JP 2001-288094 A JP 2002-193828 A JP 11-246435 A Japanese Patent No. 2729820 Japanese Patent No. 2832037 Poultry, Science, 68, 1351-1360, 1989 Murosaki et al., Biosci. Biotechnol. Biochem., 63, 373, 1999 Sakurai et al., Japanese Society of Nutrition and Food Science, 50, 133, 1997

  An object of the present invention is to provide a safe immunostimulant that has an immunostimulatory action and can be used as a food material.

  As a result of intensive studies on the effects of food materials, particularly saccharides on the immune system, the present inventors have found that certain saccharides such as D-mannose, isomaltose, laminaribiose, cellobiose have an immunostimulatory effect. The headline and the present invention were completed.

  That is, the present invention relates to D-mannose, L-arabinose, L-fucose, isomaltose, laminaribiose, cellobiose, sophorose, cordobiose, gentiobiose, 1-O-β-D-fructopyranosyl-D-fructose. Di-β-D-fructofuranose 1,2 ′: 2,1 ′ dianhydride, α-D-fructofuranose β-D-fructofuranose 1,2 ′: 2,1 ′ dianhydride, dihetero An immunostimulant comprising, as an active ingredient, at least one saccharide selected from the group consisting of lebrosan II, arabinogalactan, arabinoxylan, ethyl-β-lactoside, and glyceroyl-β-N-acetyllactosaminide provide.

  In a preferred embodiment, the immunostimulant further contains lactic acid bacteria as an active ingredient.

  In a further preferred embodiment, the immunostimulant has an action of increasing the production amount of interleukin 12 (IL-12).

  In a more preferred embodiment, the immunostimulant has an action of increasing the production amount of interferon γ (IFN-γ).

  Since the immunostimulant of the present invention enhances the production of IL-12 or IFN-γ, it has a cellular immunostimulatory effect. Therefore, it is expected to be used for cancer immunotherapy and cancer prevention. In addition, since the active ingredient of the immunostimulant of the present invention is sugar and / or lactic acid bacteria conventionally used as foods and is known to be safe, not only as pharmaceuticals but also as health foods Is available.

  The immunostimulant of the present invention contains, as an active ingredient, D-mannose, L-arabinose, L-fucose, isomaltose, laminaribiose, cellobiose, sophorose, cordobiose, gentiobiose, 1-O-β-D-fructopyra. Nosyl-D-fructose, di-β-D-fructofuranose 1,2 ′: 2,1 ′ dianhydride, α-D-fructofuranose β-D-fructofuranose 1,2 ′: 2,1 'Contains at least one saccharide selected from the group consisting of dianhydride, diheterolevrosan II, arabinogalactan, arabinoxylan, ethyl-β-lactoside, and glyceroyl-β-N-acetyllactosaminide.

  D-mannose is a 2-epimer monosaccharide of D-glucose, and as described above, it exists as a constituent component of a sugar chain in a living body as seen in a high mannose type sugar chain. It is being recognized that it plays an important role in maintaining As its physiological action, it is only known to have an action of inhibiting the establishment of Salmonella on chicken intestinal epithelial cells (Non-patent Document 1).

  L-arabinose is a monosaccharide generally contained in a heteropolysaccharide that is synthesized and secreted as an extracellular matrix by plant cells. As its physiological function, when it is ingested with sucrose, it has been reported that the digestive absorption of sucrose is suppressed and the energy utilization is reduced (Non-patent Document 3).

  L-fucose is a monosaccharide widely distributed mainly as a non-reducing terminal sugar of various carbohydrates. It is known that the role in the living body is related to the type determinant of human blood group, the uptake of serum glycoprotein into the liver, and the receptor for macrophage migration inhibitory factor.

  Isomaltose is a reducing disaccharide in which two molecules of glucose are linked by α1,6, and is present in a small amount in fermented foods. A syrupy product mainly composed of isomaltose is commercially available from Showa Sangyo Co., Ltd. As its physiological function, bifidobacteria growth activation is known.

  Laminaribiose is a reducing disaccharide in which two molecules of glucose are linked by β1,3 and is a structural unit of β1,3 glucan known to have anticancer activity. Its physiological function is hardly known.

  Cellobiose is a reducing disaccharide in which two molecules of glucose are linked by β1,4 and is a structural unit of cellulose, which is a dietary fiber, but little is known about its physiological action. Its physiological function is hardly known.

  Sophorose is a reducing disaccharide in which two molecules of glucose are linked by β1,2 and is a component of the pod of pods of legumes. Its physiological function is hardly known.

  Cozybiose is a reducing disaccharide in which two molecules of glucose are linked by α1,2 and is present in sake and koji juice. As its physiological function, it has been reported that it has hard caries, bifidobacteria growth activation action, etc. (Patent Document 9).

  Gentiobiose is a reducing disaccharide in which two molecules of glucose are linked by β1,6, and exists as a sugar component of a glycoside. This is known to have bifidobacteria growth activity.

  1-O-β-D-fructopyranosyl-D-fructose is a homodimer in which two molecules of D-fructose are linked at one location by β-fructopyranoside, and its physiological function is almost known. Not.

  Di-β-D-fructofuranose 1,2 ′: 2,1 ′ dianhydride is a 1,2 ′: 2,1 ′ anhydro sugar in which two molecules of D-furanose form are condensed with each other by 1,2 bond. is there. Little is known about its physiological function.

  α-D-fructofuranose β-D-fructofuranose 1,2 ′: 2,1 ′ dianhydride is obtained by condensing two molecules of D-fructose furanose form with 1,2 bonds to each other. 2,1 ′ anhydro sugar. Little is known about its physiological function.

  Diheterolevrosan II is a 1,2 ′: 2,1 ′ anhydro sugar in which one molecule each of pyranose form and furanose form of D-fructose is condensed with 1,2 bond to each other. Its physiological function is hardly known.

  Arabinogalactan is a polysaccharide composed of L-arabinose and galactose, and a galactose main chain bonded with β1,3 has a side chain of galactose or L-arabinose. Although arabinogalactan is used in the food field as an emulsion stabilizer, thickener, etc., its physiological function is hardly known.

  Arabinoxylan is a polysaccharide in which the side chain of L-arabinose is attached to the xylose main chain linked with β1,4. Its physiological function is hardly known.

  Ethyl-β-lactoside is produced from a lactosyl transfer reaction using an enzyme having β1,4 glucosyl bond cleavage activity, p-nitrophenyl-β-D-lactoside as a lactose donor, and ethanol as a lactose acceptor. It is a kind of lactose glycoside. Its physiological function is hardly known.

  Glyceroyl-β-N-acetyllactosaminide uses an enzyme having β1,4 glucosyl bond cleavage activity, N-acetyllactosamine as an N-acetyllactosaminyl donor, and glycerol as N-acetyllactosaminyl N-acetyllactosaminyl glycoside produced by N-acetyllactosaminyl transfer reaction as a receptor. Its physiological function is hardly known.

  These saccharides may be used alone or in admixture of two or more.

  The immunostimulant of the present invention preferably further contains lactic acid bacteria as an active ingredient. As the lactic acid bacteria, lactic acid bacteria usually used for processing foods are used, and intestinal lactic acid bacteria living in the human intestine are particularly suitable. Typically, Lactobacillus gasseri, Lactobacillus casei, Lactobacillus acidophilus, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium bifidum, Bifidobacterium breve , Bifidobacterium addressenses, Streptococcus faecalis and the like. These may be used alone or in combination of two or more.

  The immunostimulant of the present invention has the ability to produce IL-12 of macrophages or IFN-γ of lymphocytes between intestinal epithelial cells by using the saccharides alone or in combination or as a mixture with the lactic acid bacteria. Can be enhanced.

  The immunostimulant of the present invention may be a food or pharmaceutical that enhances immunity by mixing the above active ingredients with a commonly used food or food ingredient, pharmaceutical carrier or excipient by a commonly used method. it can. The food or food ingredient, pharmaceutical carrier or excipient to be used is not particularly limited and can be appropriately selected depending on the specific use of the target immunostimulator. In addition, the form of the immunostimulant is not particularly limited, and can be in the form of various solids or liquids depending on the specific application.

  When using the immunostimulant of this invention as a foodstuff, it is also possible to provide with forms, such as a seasoning, livestock meat processed goods, agricultural processed goods, and confectionery.

(Preparation example)
Lactobacillus / Gasseri ATCC 33323 was inoculated into 5 ml of MRS medium (trade name “Lactobacilli MRS Broth”, manufactured by Difco), which is a culture medium for lactic acid bacteria, and left at 32 ° C. for 24 hours. This culture solution was inoculated to 100 ml of MRS medium so as to be 1%, followed by stationary culture at 32 ° C. for 24 hours. The obtained culture solution was centrifuged at 10,000 × g for 20 minutes, and the cells were collected. The cells were suspended in physiological saline and centrifuged at 10,000 × g for 20 minutes to collect the cells. After repeating this operation three times, the cells were suspended in distilled water. This suspension was sterilized by placing it at 70 ° C. for 10 minutes, and then quickly frozen in dry ice-ethanol. This was freeze-dried to obtain 0.25 g of dried dead cells of Lactobacillus gasseri.

Example 1
A D774 medium containing 10% FBS (fetal calf serum) (hereinafter referred to as J774 cell line (available from RIKEN)), which is a mouse-derived macrophage cell line, so that the cell number is 5 × 10 ⁶ / ml Diluted simply with medium). This was seeded in a 96-well tissue culture plate at 100 μl per well and cultured in a 5% carbon dioxide incubator at 37 ° C. for 2 hours. To this was added 50 μl of a suspension obtained by dispersing Lactobacillus gasseri dried dead cells obtained in the above Preparation Example in a medium at a concentration of 4 μg / ml, and each of the sugars shown in Table 1 was added at 4 μg / ml. 50 μl of the solution contained in the medium at a concentration of ml was added per well (final concentration 1 μg / ml). As a comparison, a suspension in which Lactobacillus gasseri dried dead cells were dispersed in a medium at a concentration of 2 μg / ml or a suspension containing Lactobacillus gasseri dried dead cells and nigerose, or only the medium as a control, 100 μl was added per well. These were cultured in a 5% carbon dioxide incubator at 37 ° C. for 24 hours, and IL-12 in the culture supernatant was measured by enzyme immunoassay.

  The enzyme immunoassay for IL-12 was performed as follows. Rat anti-mouse IL-12p40 antibody (Genzyme) as a primary antibody was prepared to 4 μg / ml with PBS, and 50 μl of this primary antibody solution was added to a 96-well tissue culture plate per well and left at room temperature overnight. Rat anti-mouse IL-12p40 antibody was attached to each hole. After washing with PBS, 100 μl of PBS containing 1% bovine serum albumin (BSA) was added per well and left at room temperature for 1 hour. After washing, 50 μl of the culture supernatant was added per well and allowed to stand at room temperature for 2 hours to bind the IL-12 in the culture supernatant to the rat anti-mouse IL-12p40 antibody attached to the plate. After washing, 50 μl of a secondary antibody (detection antibody) peroxidase-labeled anti-mouse IL-12p40 antibody (Genzyme) was added and allowed to bind to IL-12 bound to the plate. After washing, 50 μl of tetramethylbenzidine (manufactured by Dako Cytomation) was added per well and reacted at room temperature for 20 minutes, and then 50 μl of 2N sulfuric acid was added per well to stop the reaction. Absorbance at 450 nm was measured with a microplate reader, and the concentration of IL-12 in the culture supernatant was determined from a standard curve prepared using recombinant mouse IL-12 (Genzyme). Table 1 shows the results.

  As can be seen from Table 1, in any sugar used in the experiment, an IL-12 concentration equal to or higher than that in the case of using nigerose known to enhance IL-12 production ability was shown. Furthermore, when only lactic acid bacteria (Lactobacillus gasseri) were used, the production amount of IL-12 was increased, but the production amount of IL-12 was further increased by simultaneously using sugar. From this, it was found that the sugar used in this experiment enhances the IL-12 production ability of macrophages, particularly when used in combination with lactic acid bacteria.

(Example 2)
The J774 cell line was cultured and cultured in the same manner as in Example 1 except that sugars having high IL-2 production ability listed in Table 2 were selected from the sugars used in Example 1 and cultured. The concentration of IL-12 in the supernatant was measured. In addition, it culture | cultivated using only a culture solution as a control, and using Lactobacillus gasseri alone as a comparison. The results are shown in Table 2.

  As can be seen from Table 2, as in the case of Example 1 above, when only lactic acid bacteria were used, the production amount of IL-12 was increased. The reproducibility of further increase was confirmed. In addition, although the production amount of IL-12 is a high value in all cases as compared with Example 1, this seems to be due to the difference in IL-12 production ability of the macrophage cell line itself used.

(Example 3)
Induction of interferon γ (IFN-γ) production in intestinal interepithelial cell lymphocytes (IEL) by isomaltoligosaccharide was examined. 10 mice (C57BL / 6, female, 4 weeks old) were given a free diet (control group) of basic diet AIN-93G (Oriental Yeast Co.), and 20% of the basic diet was made by Showa Sangyo Co., Ltd. The animals were reared in two groups: a free feed group (test group) replaced with isomalt-oligosaccharide (trade name: IMO900P, branched oligosaccharide content in the solid content of 85% or more). On day 14 of breeding, the mice were sacrificed under ether anesthesia, the small intestine was removed, and the intestinal contents were washed away with HBSS (GIBCO). After removing the Peyer's patch, the small intestine piece was chopped to 1 cm or less, Medium 199 (GIBCO) was added, and cultured with shaking at 37 ° C. for 20 minutes. After centrifugation, the supernatant was removed, RPMI1640 (manufactured by SIGMA) was added, and the mixture was again cultured with shaking at 37 ° C. for 20 minutes. Tissue masses and foreign substances were removed by filtration, and after centrifugation, the cell pellet was suspended in 44% Percoll (Pharmacia) and overlaid with 67% Percoll. Centrifugation was performed, and a layer containing intestinal epithelial cell lymphocytes (IEL) accumulated at the boundary between 44% and 67% Percoll was collected and washed twice with RPMI1640. This was used as an IEL suspension in subsequent experiments.

On the other hand, 10 μg / ml of anti-CD3 antibody was dispensed in 0.1 ml portions into a 96-well flat bottom plate and allowed to stand at 4 ° C. overnight. The plate was washed twice with HBSS and once with RPMI 1640, and 0.2 ml of IEL suspension adjusted to 2.5 × 10 ⁶ cells / ml was dispensed. This was cultured at 37 ° C. for 48 hours, and then IFN-γ contained in the culture supernatant was used in accordance with the attached instruction manual using ELISA Development Kit IFN-γ (manufactured by GT) as follows. ,It was measured. Capture antibody diluted 180-fold with PBS (Phosphate-buffered Saline) was dispensed into a 96-well flat bottom plate and allowed to stand overnight at room temperature. Remove Capture antibody, wash 3 times with Wash buffer (PBS containing 0.05% Tween20 (registered trademark)), add 0.1 ml Block Buffer (PBS containing 1% BSA, 5% Sucrose), and let stand at room temperature for 1 hour did. After removing the Block Buffer and washing 3 times with Wash buffer, 0.05 ml of the culture supernatant was added and left at room temperature for 2 hours. At the same time, a mouse IFN-γ standard solution prepared by adjusting 7 points of a 2-fold serial dilution with Reagent Diluent (Tris-buffered Saline (20 mM Trizma base, 150 mM NaCl) containing 0.1% BSA, 0.05% Tween20 (registered trademark)) was set to 0. A standard curve for quantification was prepared by adding 05 ml each. The culture supernatant and standard solution were removed, washed 3 times with Wash buffer, 0.05 ml of Detection Reagent diluted 200-fold with Reagent Diluent was added, and the mixture was allowed to stand at room temperature for 20 minutes. Detection Reagent was removed, and after washing 3 times with Wash buffer, 0.05 ml of TMB (DAKO) was added and left at room temperature for 20 minutes. The reaction was stopped by adding 0.05 ml of 2N H ₂ SO ₄ and the absorbance at 450 nm was measured. From the standard curve prepared using the mouse IFN-γ standard solution, the amount of IFN-γ in the culture supernatant was calculated. The results are shown in Table 3.

  As can be seen from Table 3, in the test group fed with the feed containing isomaltoligosaccharide, the IFN-γ production amount was significantly increased as compared with the control group.

Example 4
We investigated the life-prolonging effect of opportunistic infection model mice by ingesting isomaltoligosaccharides using an actual pathologic model system. 10 mice (C57BL / 6, female, 4 weeks old) were given a free diet (control group) of basic diet AIN-93G (Oriental Yeast Co.), and 20% of the basic diet was made by Showa Sangyo Co., Ltd. The animals were divided into two groups, a free intake group (test group) of feed replaced with isomaltoligosaccharide (trade name: IMO900P) and reared. On the 14th day, the animals were irradiated with 9 Gy of gamma rays to induce opportunistic infection, and then observed for life and death. As a result, as shown in FIG. 1, the test group tended to have a longer survival time than the control group.

  Since the immunostimulant of the present invention enhances the production of IL-12 or IFN-γ, it has a cellular immunostimulatory effect. Therefore, it is expected to be used for cancer immunotherapy and cancer prevention. Furthermore, the immunostimulant of the present invention can be used not only as a pharmaceutical product but also as a health food.

It is a figure which shows the immunostimulation effect by isomaltoligosaccharide.

Claims (4)

D-mannose, L-arabinose, L-fucose, isomaltose, laminaribiose, cellobiose, sophorose, cordobiose, gentiobiose, 1-O-β-D-fructopyranosyl-D-fructose, di-β-D -Fructofuranose 1,2 ': 2,1' dianhydride, α-D-fructofuranose β-D-fructofuranose 1,2 ': 2,1' dianhydride, diheterolevrosan II, arabino An immunostimulant comprising as an active ingredient at least one saccharide selected from the group consisting of galactan, arabinoxylan, ethyl-β-lactoside, and glyceroyl-β-N-acetyllactosaminide and lactic acid bacteria . The immunostimulant according to claim 1, wherein the lactic acid bacterium is Lactobacillus gasseri .   The immunostimulant of Claim 1 or 2 which has the effect | action which raises the production amount of the interleukin 12.   The immunostimulator of Claim 1 or 2 which has the effect | action which raises the production amount of interferon (gamma).

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