JP7008301B2 - Method for evaluating immunostimulatory activity of orally ingested materials - Google Patents

Description

The present invention relates to a method for evaluating the immunostimulatory activity of an orally ingested material using an isomaltose heated product, which is a substance having an action of inducing the production of granulocyte colony stimulating factor (G-CSF) in the gastrointestinal tract, as an index .

The body of animals, including humans, is constantly exposed to the risk of illness due to exogenous microorganisms such as bacteria and viruses, and endogenous foreign body self-substances such as tumor cells. It has an immune system as an intrinsic defense mechanism to combat the disease. If the function of this immune system can be strengthened, not only is it difficult to suffer from a disease, but it can be expected that the affected disease will be alleviated or treated by autoimmune function.

Therefore, various studies have been conducted on strengthening the immune function of a living body, that is, activating the activation of immunity. For example, Patent Document 1 discloses an immunostimulatory substance containing ascophyllan extracted from ascophyllum nodosum as an active ingredient. Patent Document 2 discloses that the liquid-liquid solvent extraction fraction of seaweed belonging to the genus Gracilaria has a macrophage activating effect. Patent Document 3 discloses that an acidic polysaccharide extracted from a seaweed belonging to red algae is enzymatically treated and has a macrophage activating effect. Patent Document 4 discloses that the Porphyran enzymatic degradation product has an ability to induce the production of activated interleukin 12. Patent Document 5 discloses that porphyran, which is a water-soluble fraction derived from Amanori, improves the ability of mesenteric lymph node lymphocytes to produce IgA and the serum IgA concentration. Patent Document 6 discloses that an alginic acid oligomer, which is an alginic acid decomposition product, acts on human terminal blood leukocytes to induce the production of tumor necrosis factor. Patent Document 7 describes unsaturated fatty acids derived from seaweed, 4,7,10,13-hexadecatetraenoic acid, 6,9,12,15-octadecatetraenoic acid, 5,8,11,14, It is disclosed that 17-eicosapentaenoic acid, 9,12,15-octadecatorienic acid activates eosinophils.

Japanese Unexamined Patent Publication No. 2008-120707 Japanese Unexamined Patent Publication No. 5-139988 Japanese Unexamined Patent Publication No. 6-256208 JP-A-2002-193828 Japanese Unexamined Patent Publication No. 2005-126429 Japanese Unexamined Patent Publication No. 2005-145885 Japanese Unexamined Patent Publication No. 2005-23028

By the way, as one of the cytokines produced in the human body having an action of enhancing immunity, there is Granulocyte Colony Stimulating Factor (G-CSF). G-CSF acts on granulocyte stem cells in the bone marrow to promote differentiation, promotes the outflow of the produced granulocytes from the bone marrow, and has an effect of enhancing the function of neutrophils. The recombinant human G-CSF preparation is used for neutropenia caused by cancer chemotherapy and neutropenia associated with aplastic anemia. For example, for leukopenia seen during anticancer drug treatment, G-CSF etc. are administered to improve clinical effects, such as leukocytosis during infection, leukocytosis after cancer chemotherapy or aplastic anemia, etc. The effect of G-CSF in G-CSF is being studied, but high cost and technology are required for production (purification).

Therefore, as a result of diligent studies by the present inventors, a new substance having an action of inducing the production of granulocyte colony stimulating factor (G-CSF) in the gastrointestinal tract from easily available raw materials has never existed before. It was found that the present invention could be obtained, and the present invention was completed. That is, the present invention comprises an immunostimulatory active composition and an immunostimulatory activator containing a substance having an action of inducing the production of G-CSF in the gastrointestinal tract as an active ingredient, a method for producing the substance, and immunostimulatory of an orally ingested material. It provides an activity evaluation method.

The following inventions are proposed.
( 1 ) An orally ingested material that measures the content of isomaltose in the orally ingested material and evaluates the degree of immunostimulatory activity of the heated product obtained by heating the orally ingested material to 150 to 200 ° C. using the content as an index. Immune activation activity evaluation method.

Oral ingestion of isomaltose heated products induces and promotes the production of G-CSF in the gastrointestinal tract. As a result, the activation of immunity in the gastrointestinal tract is activated, and treatment or prevention of various diseases caused by the gastrointestinal tract can be expected. In the present invention, the "action of inducing G-CSF production from the gastrointestinal tract" means "enhancing the function of the gastrointestinal tract and improving energy metabolism" in traditional medicine.

Some orally ingested ingredients such as crude drugs and foodstuffs originally contain isomaltose. Therefore, by measuring the isomaltose content in the orally ingested material, it is possible to predict in advance the degree of contribution to the immunostimulatory activity by G-CSF when the orally ingested material is ingested by cooking or the like. can.

It is a bar graph which shows the degree of activation activity of each sugar. It is a bar graph which shows the activity effect by temperature. It is a graph which shows the relationship between the content of isomaltose and the immunostimulatory activity effect. It is a graph which shows the activation activity effect of an orally ingested material. It is the effect confirmation test result of natural honey.

The active ingredient of the immunostimulatory active composition or the immunostimulatory active agent of the present invention is an isomaltose heated product. The isomaltose heating product is an action of inducing the production of granulocyte colony-stimulating factor (G-CSF), which is a cytokine having an action of enhancing immunity, from gastrointestinal epithelial cells by heating isomaltose at a predetermined temperature. It is a substance that has changed to a substance with. The gastrointestinal tract here means the small intestine (duodenum, jejunum, ileum) and the large intestine (cecum, colon, rectum).

The substance changed from the isomaltose is a polymer compound having a mass average molecular weight of about 840,000, although the details have not been determined. When hydrolyzed with trifluoroacetic acid to investigate the characteristics of this polymer compound, it contained a single saccharide other than glucose and fructose.

The isomaltose heating product can stimulate either or both of the Toll-like receptors (TLRs) TLR2, TLR4. Toll-like receptors are receptor proteins on the surface of cells responsible for the immune system, and have the effect of recognizing various pathogens and activating innate immunity. Types 1 to 11 are known, and type 2 recognizes lipoteichoic acid present in the cell wall of Gram-positive bacteria, and type 4 recognizes lipoteichoic acid present in the cell wall of Gram-negative bacteria.

The isomaltose heating product is obtained by heating the isomaltose at 150 to 200 ° C. When the heating temperature is low, the substance does not change and the effect of activating immunity cannot be obtained. On the other hand, if the heating temperature is too high, carbonization of isomaltose progresses and the immunostimulatory activity effect is ineffective. In the range of 150 to 200 ° C., the heating temperature is preferably as high as possible. This is because the heating time can be shortened. For example, when heating at 150 ° C., a heating time of 4 hours or more is required, but at 200 ° C., heating for 15 minutes or more may be sufficient. The heating temperature is preferably 170 to 190 ° C. in order to obtain a high activation activity effect in a short time as possible and to prevent carbonization.

The immunostimulatory active composition containing the isomaltose heated product of the present invention as an active ingredient is an oral composition in the form of suspension, emulsion, syrup, extract, liquid or the like as it is or mixed with other additives. Can be. Alternatively, these immunostimulatory active compositions may be appropriately processed by a conventional method to obtain oral immunostimulatory activators such as powders, fine granules, granules, tablets and capsules.

Excipients to be added to the immunostimulatory active composition or the immunostimulatory activator include gelatin, sodium alginate, starch, corn starch, mannitt, carboxymethyl cellulose, dextrin, polyvinylpyrrolidone, crystalline cellulose, soy lecithin, fatty acid ester and the like. Agents, binders, disintegrants, surfactants, lubricants, fluidity promoters, diluents, preservatives, colorants, fragrances, lecithins, stabilizers, moisturizers, preservatives, antioxidants, etc. Can be used.

In addition, in order to supplement other nutrients as well as activating immunity, sweeteners such as sugar, fructose, glucose, fructose-dextrose liquid sugar, aspartame, and stevia, acidulants such as citric acid, apple acid, and ascorbic acid, and vitamin A group. : Retinol, retinal (vitamin A1), dehydroretinal (vitamin A2), carotene, lycopene (provitamin A), vitamin B group: thiamine hydrochloride, thiamine sulfate (vitamin B1), riboflavin (vitamin B2), pyridoxin (vitamin) B6), cyanocobalamine (vitamin B12), folic acid, nicotinic acid, pantothenic acid, biotin, choline, inositol, vitamin C group: vitamin C acid or its derivative, vitamin D group: ergocalciferol (vitamin D2), kore Calciferol (vitamin D3), dihydrotaxterol, vitamin E group: vitamin E or its derivatives, ubiquinones, vitamin K group: phytonadione (vitamin K1), menaquinone (vitamin K2), menadione (vitamin K3), menadiol (vitamin K4) ), Other essential fatty acids (vitamin F), carnitine, ferulic acid, Γ-orizanol, orotoic acid, vitamin Ps (rutin, eriocitrin, hesperidin), vitamins such as vitamin U, sodium, potassium, calcium, magnesium, Minerals such as iron, zinc, copper, phosphorus, manganese, iodine, valine, leucine, isoleucine, threonine, methionine, phenylalanine, tryptophan, lysine, glycine, alanine, asparagine, glutamine, serine, cysteine, cystine, tyrosine, proline, Hydroxyproline, aspartic acid, glutamic acid, hydroxylysine, arginine, ornithine, histidine, etc., their citrates, amino acids such as pyrrolidone carboxylic acid, myristic acid, palmitic acid, stearic acid, palmitreic acid, oleic acid, linoleol It is also possible to add one or more selected from fatty acids such as acid, linolenic acid, docosapentaenoic acid (DPA), tocosahexaenoic acid (DHA), eikosapentaenoic acid (EPA), lipids, salts, proteins and the like. ..

Furthermore, in order to improve palatability, it assists in the absorption of thickening polysaccharides (trommy components) such as xanthan gum, carrageenan, guar gum, arabic gum, tragant gum, karaya gum, gutti gum, pullulan, and mineral absorption such as casein, casein sodium, and casein calcium. It is also possible to add one or more selected from sugar alcohols such as agents, sorbitol, xylitol, raffinose, glucose and martitol.

Further, by adding an isomaltose heated product to a known food / beverage / herb / herbal medicine or the like, it is possible to obtain a functional food or a functional beverage that activates immunity.

Foods include fresh cooked foods such as salads; cooked foods such as steaks, pizzas and hamburgers; fried cooked foods such as fried vegetables; vegetables such as tomatoes, peppers, celery, niggauri, carrots, potatoes and asparagus. Cooked products made from these vegetables; cookies, bread, biscuits, dried bread, cakes, rice cakes, sheep rice, pudding, jelly, ice cream, chewing gum, crackers, chips, chocolate and sweets such as candy; udon, pasta, and buckwheat Noodles such as; fish paste products such as kamaboko, ham, and fish sausage; dairy products such as cheese, cream, and butter; miso, soy sauce, dressing, ketchup, mayonnaise, soup base, noodle soup, curry powder, mirin, ru , Seasoning spices and other seasonings; soy foods such as tofu; konjac; and supplements. Foods preferably include roux (eg, for curry or stew), seasoning spices, supplements and the like.

Beverages include, for example, coffee beverages; cocoa beverages; vegetable juices obtained from the above vegetables; fruit juice beverages such as grapefruit juice, orange juice, grape juice, and lemon juice; tea beverages such as green tea, tea, roasted tea, and oolong tea; Alcoholic beverages such as beer, wine (red wine, white wine, or sparkling wine, etc.), sake, plum wine, sparkling liquor, whiskey, brandy, shochu, lamb, gin, and liqueurs; milk beverages; soy milk beverages; liquid foods; and Sports drinks and the like can be mentioned.

Herbs include, for example, artichoke, anise, alfalfa, aloe, angelica, evening primrose, echinacea, elderflower, oat wheat, oregano, chamomile, galana, cardamon, caraway, cranberry, cloves, goldenseal, gotuccola, coriander, cinnamon, Jasmine, Ginger, Ginseng, Spearmint, St. Jones Wort, Thyme, Tea, Dill, Hibiscus, Basil, Passion Flower, Valerian, Bilberry, Fennel, Peppermint, Bergamot, Hop, Bolizi, Maca, Mate, Marigold, Mint, Eucalyptus, Yohinbe , Lavender, rosehip, rosemary, licorice, louis boss, lemon balm, lemongrass, lemon peel and the like.

Herbal medicines include Akyo, Inyoukaku, Ougi, Osei, Kaikujin, Kashu, Eucommia ulmoides, Eucommia ulmoides, and Kanrensou.旱 lotus grass), Kiban (turtle plate), Kibankyo (turtle plate glue), Kyuushi (韮子), Gyokuchiku (Tamatake), Kinoshi (Kinsakurako), Kukoshi (枸杞子), Kuseki (Kuseki), Koui ( Glue), Gokai (Gourmet), Kokuzu (Black Bean), Kotsusaiho (Bone Crushing Supplement), Kotouniku (Gourmet Meat), Sesame (Goma), Koroha (Koroba), Sayo (Chainyang), Sanyaku (Yamayaku) ), Jio (Jihuang), Shikasha (Shikawasha), Shakuyaku (Glue), Shajin (Sasan), Joteishi (Female Sadako), Zion (Shien), Seiyoujin (Western Ginseng, Flower Flag Ginseng), Sekkoku (Ishibashi), Sempukuka (returned flower), Senbo (Senkyo), Sokisei (Mulberry parasite), Soujin (Kuwaso), Souhakuhi (Kuwashirohide), Zokudan (Continued), Taishijin (Taishijin), Taiso (Daiko), Tenmondou (Tenmon Fuyu), Touki (Toki), Toushitsuri (Kanshitsu), Toujin (Party), Tochu Kasou (Fuyu-mushi Natsukusa), Toshishi (Eucommia ulmoides), Tochu (Tochu), Nanshajin (Nanshajin), Nikujuyo (Nikujuyo) ), Carrot (carrot), Bakumondou (wheat gate winter), Hagekiten (Baekiten), Byakugo (yuri), Byakujutsu (white 朮), Byakuzen (white front), Byakubu (hundred parts), Biwayou (枇杷 葉), Bekko (Tortoiseshell), Beckokyo (Glue), Hokotsushi (Bone fat), Maou (Mao), Yakuchinin (Masutomohito), Ryugannik (Ryu-eye meat), Rokujo (Deer mushroom), Rokkaku (Deer horn), Rokkakukyo (Deer horn) Glue), rock squirrel (deer horn frost), etc. can be mentioned.

The content of the immunostimulatory active composition, the immunostimulatory activator, and the isomaltose heated product in the functional food or the functional beverage is not particularly limited, and is 1 to 100% by weight, preferably 5 to 90% by weight, more preferably. May be 10 to 80% by weight. The higher the content of the isomaltose heated product, the higher the immunostimulatory activity function.

Further, if it is an orally ingested material that originally contains isomaltose, it can be directly made into a functional food or the like by heating it at 150 to 200 ° C. The immunostimulatory activity effect at this time is also proportional to the content of isomaltose originally contained. Therefore, by measuring the isomaltose content of the orally ingested material, the degree of effectiveness as a functional food for immunostimulatory activity can be estimated and evaluated.

Typical oral intake materials that originally contain isomaltose include, for example, honey, fermented foods (miso, soy sauce, sake, lees, amazake, vinegar, cheese, yogurt), syrup, oligosaccharides, and the like. .. Among them, honey, syrup, and oligosaccharides that are easily available and have a relatively high content of isomaltose are preferable, and isomaltoligosaccharide is more preferable as the oligosaccharide.

(Randomized controlled trials by sugar)
First, we focused on sugar as an active ingredient of activation activity, and compared and examined the activity effect for each type of sugar. Fructose, glucose, sucrose, turanose, maltose, trehalose, isomaltose, and melezitose were used as sugars, and the degree of activation activity was measured by the following methods.

In a pre-weighed test tube (15 mL), 100 μL of an aqueous solution of various saccharides (100 mg / mL) was placed and dried at 80 ° C. overnight. The test tube containing the sample was placed upright in a thermostat heated to 180 ° C., and heated for 60 minutes when the temperature rose to 180 ° C. again. The weight of the test tube after heating was measured, and the amount of decrease in weight after heating was calculated. Then, 4 mL of hot water was added to the test tube, and the heated product was melted by standing in boiling water and freeze-dried. Heated product Water was added so as to be 100 mg / mL of hot water, and the mixture was stored at −20 ° C. until use.

MCE301 cells derived from mouse colonic epithelium (provided by Professor Keiaki Tabuchi, Life Science Research Center, University of Toyama) were used for the immunostimulatory activity evaluation test. The culture was carried out using a flask having a bottom area of 75 cm ² under the conditions of 37 ° C. and 5% CO ₂ . Dulbecco's modified Eagle medium / Ham's F-12 (1: 1) (DMEM) containing 5% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA), 100 U / mL penicillin + 100 μg / mL streptomycin. / F-12) When the cells have grown to 80-90% confluent using medium (Sigma) as the growth medium, use 0.25% trypsin (Sigma) /0.02% EDTA · 2Na to grow the cells. It was peeled off and subcultured.

MCE301 cells were seeded on 96-well plates at a density of 4 × 10 ⁴ cells / well and incubated at 37 ° C. and 5% CO ₂ for 24 hours. The cells were replaced with serum-free medium and cultured at 39 ° C. and 5% CO ₂ for 72 hours. The cells were replaced with media containing various samples and cultured at 39 ° C. and 5% CO ₂ for 24 hours. Then, the medium was transferred at 50 μL / well to another 96-well plate coated with poly-L-lysine (Sigma) and dried at 38 ° C.

The plate was washed with phosphate buffered saline (PBS, 0.15M, pH 7.4), 60 μL / well of Block Ace (DS Pharma Biomedical, Osaka) was added, and the mixture was allowed to stand at room temperature for 2 hours. Rabbit anti-G-CSF antibody (Boster Biological Technology, Pleasanton, CA, USA) was diluted 500-fold with a solution of Block Ace diluted 10-fold with PBS, 50 μL / well was added, and the mixture was allowed to stand at room temperature for 2 hours. .. After washing 5 times with PBS, donkey anti-rabbit IgG (HRP) (ab205722, Abcam, Cambridge, UK) was added with 50 μL / well diluted 2000 times with a solution of Block Ace diluted 10 times with PBS. It was allowed to stand at room temperature for 2 hours. After washing 5 times with PBS, ABTS Solution (Roche Applied Science, Upper Bavaria, Germany) was added, and the absorbance at 405 nm was measured with a microplate reader. It has been confirmed that the G-CSF concentration correlates with the absorbance at 405 nm by this method.

The test results are shown in FIG. The zero index in each figure means a medium containing no sugar sample, and is used as an index having zero immunostimulatory activity. On the other hand, the positive index (positive control) is a sample using Pokeweed mitogen (PWM, 100 μg / mL, Sigma). Pokeweed has been known to have an activity of stimulating immune system cells, and is used as an index for comparing the positive effects of the present invention.

As is clear from the results of FIG. 1, it was confirmed that only the isomaltose heated product has a high absorbance at 405 nm, that is, the G-CSF concentration, and that other sugars do not have the effect of inducing the production of G-CSF in the gastrointestinal tract. rice field.

(Evaluation of heating temperature)
Therefore, the heating temperature of isomaltose was variously changed, and the temperature at which the active effect was exhibited was evaluated. Specifically, the absorbance at 405 nm was measured by the same method as in the above sugar-specific comparative test except that the isomaltose heated product water (hereinafter referred to as a sample) was heated at a concentration of 250 μg / mL under the conditions shown in Table 1. did. The results are shown in FIG. Since each sample was conducted on a different schedule in this test, the value of the zero index in each sample result was set to 0 and the value of the positive index was set to 100 so that the degree of activation activity could be directly compared, and the absorbance was the titer (%). ) Is converted.

As is clear from the results of FIG. 2, it was found that G-CSF was hardly produced even when isomal and sose were heated at 120 ° C., and the activation activity effect could not be obtained. On the other hand, it was found that the activation activity effect can be obtained by heating at 150 ° C. or higher. In particular, the G-CSF concentration was high when heated at 180 ° C. From this, it was found that it is preferable to heat at 170 to 190 ° C.

(Concentration-dependent evaluation)
Subsequently, the relationship between the isomaltose content and the immunostimulatory activity was evaluated. Specifically, the absorbance at 405 nm was measured by the same method as in the above sugar-specific comparative test except that each sample had the concentration shown in Table 2. The results are shown in FIG.

From the results shown in FIG. 3, it was confirmed that the content of isomaltose and the immunostimulatory activity effect are in a proportional relationship.

(Oral ingestion material test)
Subsequently, the immunostimulatory activity effect of the foodstuff (orally ingested material) originally containing isomaltose was confirmed. Specifically, simulated honey having the composition shown in Table 3 was prepared, and the absorbance at 405 nm was measured by the same method as in the above sugar-specific comparative test. The results are shown in FIG. The numerical values shown in Table 3 are% by weight.

From the results shown in FIG. 4, it was confirmed that the immunostimulatory activity effect can be obtained even if the orally ingested material containing isomaltose is heated as it is. On the other hand, it was also confirmed that the immunostimulatory activity effect could not be obtained with the orally ingested material containing no isomaltose.

(Isomaltose content dependence test)
Therefore, various natural honeys (natural honeys derived from acacia, astragalus propinata, leonurus japonicum, longan, bodaiju, wolfberry, astragalus, sunflower, and jujube) were used to evaluate their activation and activity effects. The results are shown in FIG. The evaluation method is the same as the above-mentioned sugar-specific comparative test.

The sugar content of each natural honey was measured as follows. Each honey hot water extract was prepared at 100 mg / mL with 30% acetonitrile, filtered through a 0.45 μm filter, and measured under the following HPLC conditions.
Column: COSMOSIL Sugar-D column 4.6 x 250 mm (Nacalai Tesque)
Mobile phase: water: acetonitrile (15:85); 1.0 mL / min
Temperature: 35 ° C
Detection: Differential Refractometer Sample Injection Volume: 10 μL

From the results shown in FIG. 5, it was confirmed that the higher the content of isomaltose, the higher the immunostimulatory activity effect. From this, it was found that the degree of effectiveness as a functional food for immunostimulatory activity can be estimated and evaluated by measuring the isomaltose content of the orally ingested material.

Claims (1)

The content of isomaltose in the orally ingested material is measured, and the degree of immunostimulatory activity of the heated product obtained by heating the orally ingested material to 150 to 200 ° C. is evaluated using the content as an index. Activity evaluation method.

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