WO2011108275A1 - イムノグロブリンa分泌促進剤 - Google Patents
イムノグロブリンa分泌促進剤 Download PDFInfo
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- WO2011108275A1 WO2011108275A1 PCT/JP2011/001261 JP2011001261W WO2011108275A1 WO 2011108275 A1 WO2011108275 A1 WO 2011108275A1 JP 2011001261 W JP2011001261 W JP 2011001261W WO 2011108275 A1 WO2011108275 A1 WO 2011108275A1
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- Prior art keywords
- iga
- extract
- iga antibody
- secretion
- antibody
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- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 239000011975 tartaric acid Substances 0.000 description 1
- 235000002906 tartaric acid Nutrition 0.000 description 1
- 239000000606 toothpaste Substances 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
- 235000008939 whole milk Nutrition 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
- A23G1/00—Cocoa; Cocoa products, e.g. chocolate; Substitutes therefor
- A23G1/30—Cocoa products, e.g. chocolate; Substitutes therefor
- A23G1/32—Cocoa products, e.g. chocolate; Substitutes therefor characterised by the composition containing organic or inorganic compounds
- A23G1/48—Cocoa products, e.g. chocolate; Substitutes therefor characterised by the composition containing organic or inorganic compounds containing plants or parts thereof, e.g. fruits, seeds, extracts
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
- A23G3/00—Sweetmeats; Confectionery; Marzipan; Coated or filled products
- A23G3/34—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof
- A23G3/36—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds
- A23G3/48—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds containing plants or parts thereof, e.g. fruits, seeds, extracts
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
- A23G4/00—Chewing gum
- A23G4/06—Chewing gum characterised by the composition containing organic or inorganic compounds
- A23G4/068—Chewing gum characterised by the composition containing organic or inorganic compounds containing plants or parts thereof, e.g. fruits, seeds, extracts
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
- A23G9/00—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor
- A23G9/32—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor characterised by the composition containing organic or inorganic compounds
- A23G9/42—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor characterised by the composition containing organic or inorganic compounds containing plants or parts thereof, e.g. fruits, seeds, extracts
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/52—Adding ingredients
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
- A23P10/00—Shaping or working of foodstuffs characterised by the products
- A23P10/20—Agglomerating; Granulating; Tabletting
- A23P10/28—Tabletting; Making food bars by compression of a dry powdered mixture
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
- A23P10/00—Shaping or working of foodstuffs characterised by the products
- A23P10/30—Encapsulation of particles, e.g. foodstuff additives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/66—Papaveraceae (Poppy family), e.g. bloodroot
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/08—Antiallergic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Definitions
- the present invention relates to an IgA secretion promoter that can be expected to prevent infections such as bacteria and viruses and prevent allergies, and in particular, to a composition that can be expected to promote IgA secretion in the upper respiratory tract.
- a powerful defense system called mucosal immunity has been established on the surface of the living body such as the oral cavity, respiratory tract, and digestive tract that contacts the outside world to prevent the entry of viruses and bacteria from the outside world into the living body.
- mucus On the mucosal surface, mucus is constantly secreted from the salivary glands and mucous glands, etc., physically preventing the invasion of viruses and bacteria, and protecting the living body from external enemies by the action of antibodies, antibacterial peptides, enzymes, etc. present in the mucus.
- Immunoglobulin A (IgA) antibody plays a central role in this defense system, and has actions such as removal of viruses and bacteria and prevention of adhesion to mucosal surfaces (Non-patent Document 1). ).
- Non-patent Document 2 it has been reported that when IgA antibodies specific to causative bacteria of otitis media are increased in animal experiments, infection of causative bacteria is suppressed. In addition, it has been reported that when an IgA antibody specific to a certain cold-causing virus is administered into a human nasal cavity, infection of the virus is suppressed (Non-patent Document 3).
- the IgA antibody is also considered to prevent foreign antigens that cause allergic reactions from entering the living body through the mucosa (non Patent Document 4).
- IgA antibody if the secretion of IgA antibody can be promoted, it is considered effective for the prevention of infectious diseases such as bacteria and viruses and the prevention of allergies.
- Patent Documents 1 to 4 Various lactic acid bacteria and basidiomycetes (Patent Documents 1 to 4), polysaccharides and oligosaccharides (Patent Documents 5 to 9 and Non-Patent Document 5) have been disclosed so far as having the effect of promoting secretion of IgA antibodies. However, most are related to the production of IgA antibody in the intestine, and there are few things related to the IgA secretion promoting action and the infection suppressing action in respiratory organs such as the oral cavity, nasal cavity and respiratory tract (Patent Document 2, Patent Document). 10).
- an object of the present invention is to provide a pharmaceutical composition having an IgA secretion promoting action not only in the intestine but also in the oral cavity and the respiratory tract.
- dairy poppy extract has an IgA secretion promoting action, and thus completed the present invention.
- the corn poppy extract of the present invention has not only an IgA secretion promoting effect in the intestine but also an IgA secretion promoting action in the oral cavity and the respiratory tract, and therefore, ingesting this suppresses infection with viruses and bacteria, Moreover, the occurrence of allergies can be prevented.
- Daisies (scientific name: Papaver rhoeas L.), whose IgA secretion-promoting action was recognized in the present invention, is a plant belonging to the family Papaveraceae, and is also known as a beautiful plant. It is said to have sedation, hypnosis, and analgesia, and is used as an antitussive and sedative.
- the poppy extract used in the present invention can extract daisies flowers and leaves / stems, preferably daisies flowers, using water or a mixture of water and a hydrophilic organic solvent. is there.
- the extraction method and the type of solvent are not particularly limited.
- the solvent in addition to water, alcohols such as ethanol, methanol, propanol, glycerin and propylene glycol, organic solvents such as ether, acetone, ethyl acetate, chloroform and hexane, or a combination thereof can be used. However, it is desirable to extract using water, ethanol, or a mixed solution thereof from the viewpoint of safety.
- extraction can be performed at any of high temperature, room temperature, and low temperature, but it is preferably at 40 to 90 ° C. for about 0.5 to 5 hours.
- the extract can be used as it is in the form of a solution, but it can be used after it has been filtered and the extraction solvent has been distilled off, and is then made into a powder by vacuum drying or freeze drying, or concentrated. it can. Further, those obtained by fractionating and purifying these extracts by organic solvent fractionation, column chromatography or the like can also be used. Furthermore, purification treatment such as deodorization and decolorization may be added.
- the content of corn poppy extract is not particularly limited, but is preferably 0.01% by weight or more, preferably 0.1 to 90% by weight in terms of dry weight with respect to the food or drink or the preparation. .
- the pharmaceutical composition containing the corn poppy extract of the present invention can be used, for example, as a pharmaceutical, a quasi-drug or a food and drink.
- oral dosage forms include tablets, capsules, powders, syrups, and drinks.
- oral dosage forms such as tablets, capsules, drinks and the like.
- food and drink in addition to beverages such as drinks, foods such as candy, rice crackers, and cookies, preference items such as tobacco and chewing gum, feeds and feeds, or cosmetics such as mouthwashes and toothpastes, and the like.
- it may contain one or more pharmacologically acceptable carriers and, if necessary, other active ingredients for treatment.
- it contains excipients, binders, disintegrants, lubricants, dispersants, surfactants, plasticizers, suspending agents, emulsifiers, diluents, buffers, antioxidants, bacterial inhibitors, etc. May be.
- the components other than the corn poppy extract are not limited to the above, and the present invention may optionally contain any known components.
- the pharmaceutical, quasi-drug, food and drink, etc. containing the corn poppy extract of the present invention can be produced by any method known in the respective technical fields.
- corn poppy extract may be added by any known method.
- the pharmaceutical composition containing the corn poppy extract of the present invention can be used not only for humans but also for animals other than humans (hereinafter abbreviated as non-human animals).
- non-human animals include non-human animals such as mammals, reptiles, amphibians and fish.
- RPMI1620 medium containing 10% FCS, 50 ⁇ M 2-mercaptoethanol, 1 mM sodium pyruvate, 100 U / ml penicillin, 100 ⁇ g / ml streptomycin was used.
- 0.05 ml each of a lymphocyte cell suspension and a culture medium in which the sample is dissolved are added at 37 ° C. and 5% CO 2 .
- Each well was washed with PBST, and 25 ng of peroxidase-conjugated anti-mouse IgA antibody was added to each well and incubated at room temperature for 1 hour, and then a peroxidase substrate was added and allowed to react for 2 minutes. 0.5N hydrochloric acid was added to stop the reaction, and the absorbance at 450 nm was measured.
- a calibration curve was prepared using mouse IgA antibody as a standard substance, and the amount of IgA antibody was measured.
- the results are as shown in Table 1.
- the amount of IgA antibody in the medium when the daisy (flower) hot water extract was added to the medium at a concentration of 100 ppm was 2.6 times that when nothing was added.
- mice-1 A test feed was prepared by mixing 5% (w / w) of daisy (flower) hot water extract with normal feed (MF, oriental yeast).
- MF normal feed
- mice Six 7-week-old mice (BALB / c strain) were divided into a control group and a test group (3 mice for each group).
- Feces are collected every week from the start of breeding, suspended in a phosphate buffer so as to have a concentration of 100 mg / ml, centrifuged, and the total IgA antibody amount in the supernatant is the same as in Example 2. The measurement was performed by sandwich ELISA.
- mice-2 A test feed was prepared by mixing 5% (w / w) of daisy (flower) hot water extract with normal feed (MF, oriental yeast). Twelve 7-week-old mice (BALB / c strain) were divided into a control group and a test group (6 mice in each group), and the control group was fed with a normal feed, and the test group was fed with a test feed freely. At 4 weeks, 50 ⁇ g of phosphorylcholine-keyhole limpet hemocyanin (PC-KLH, an antigen common to bacteria) was administered intranasally to immunize.
- PC-KLH phosphorylcholine-keyhole limpet hemocyanin
- the total IgA antibody amount and the PC-specific IgA antibody titer were measured by sandwich ELISA, and the number of PC-specific IgA antibody-producing cells was measured by the enzyme-linked immunospot (ELISPOT) method. Since the number of lamina limba lymphocyte cells that can be collected from each animal is very small, the number of IgA antibody-producing cells was measured in triplicate by combining three cells.
- ELISPOT enzyme-linked immunospot
- IgA Antibody Amount by Sandwich ELISA Method The total IgA antibody amount was measured by a sandwich ELISA method as in Example 2. The amount of the PC-specific IgA antibody was measured by sandwich ELISA in the same manner as in Example 2 except that PC-BSA was used instead of the anti-mouse immunoglobulin antibody that was first adsorbed on the ELISA plate.
- a chromogenic substrate (3-amino-9-ethylcarbazole) solution was added, and color was allowed to develop for 30 minutes at room temperature under light-shielding conditions. After washing with water and drying, the spots were counted as antibody-producing cells under a stereomicroscope.
- mice-3 A test feed was prepared by mixing 5% (w / w) of daisy (flower) hot water extract with normal feed (MF, oriental yeast). Twenty 7-week-old mice (BALB / c strain) were divided into a control group and a test group (10 mice in each group), and in the same manner as in Example 4, the total amount of IgA antibody in nasal mucus and PC-specific IgA antibody titer The total amount of IgA antibody in stool was measured.
- mice Seven-week-old mice (BALB / c system) were divided into a control group and a treatment group as 4 mice per group, and were bred with a normal feed (MF, oriental yeast). From the start of the breeding, dairy (flower) hot water extract was sublingually administered as an aqueous solution at a dose of 4 mg / day from the beginning of the breeding, and the control group was sublingually administered only the same amount of water.
- the results are as shown in Tables 6 and 7.
- the total IgA amount of nasal mucus and the total number of IgA-producing cells are higher in the administration group, and IgA secretion in the respiratory system is administered by sublingual administration of dairy extract. It was thought that IgA producing cells increased.
- mice Seven-week-old mice (BALB / c strain) were divided into a control group and a treatment group as 15 mice per group, and were bred with a normal diet (MF, oriental yeast). From the start of the breeding, dairy (flower) hot water extract was sublingually administered as an aqueous solution at a dose of 4 mg / day from the beginning of the breeding, and the control group was sublingually administered only the same amount of water.
- the total IgA antibody amount and PC-specific IgA antibody titer were measured by sandwich ELISA as in Example 2, and the number of IgA antibody-producing cells was measured by the enzyme-linked immunopot (ELISPOT) method as in Example 4. . Since the number of lamina limba lymphocyte cells that can be collected from each animal is very small, three cells were collected and the number of IgA antibody-producing cells was measured in duplicate.
- ELISPOT enzyme-linked immunopot
- mice Seven-week-old mice (BALB / c strain) were divided into a control group and a treatment group as 6 mice per group, and were bred with a normal feed (MF, oriental yeast). From the start of breeding, the administration group was nasally administered with a hot water extract of daisy (flower) at a dose of 2 mg / day every day as an aqueous solution, and the control group was administered nasally with only the same amount of water.
- mice-2 Five to six-month-old mice (BALB / c strain) were divided into a control group and a treatment group as five mice per group, and were bred with a normal diet (MF, oriental yeast). From the start of breeding, the administration group was nasally administered with a hot water extract of daisy (flower) at a dose of 2 mg / day every day as an aqueous solution, and the control group was administered nasally with only the same amount of water.
- MF normal diet
- Immunized with 50 ⁇ g of PC-KLH by nasal administration at 4 weeks, followed by nasal immunization every week, and 1 week after the third nasal immunization, nasal mucus and lamina propria lymphocyte cells The total IgA antibody amount and the total number of IgA antibody-producing cells were measured.
- the total IgA antibody amount was measured by the sandwich ELISA method as in Example 2, and the number of IgA antibody-producing cells was measured by the enzyme-linked immunopot (ELISPOT) method as in Example 4. Since the number of lamina intestinal lymphocyte cells that can be collected from each animal is very small, 2 to 3 cells were collected and the number of IgA antibody-producing cells was measured in duplicate.
- Tablets were prepared according to the following formulation. Lactose 54.5% Corn starch 38.5 Magnesium stearate 2.0 Daisy extract of Example 1 5.0 100.0%
- Capsules were prepared according to the following formulation. Lactose 8.0% Magnesium stearate 2.0 Daisy extract of Example 1 90.0 100.0%
- a powder was prepared according to the following formulation. Lactose 40.0% Potato starch 15.0 Daisy extract of Example 1 45.0 100.0%
- Chewing gum was prepared according to the following formulation. Gum base 20.0% Sugar 52.0 Glucose 14.0 Minamata 13.0 Fragrance 0.5 Daisy extract of Example 1 0.5 100.0%
- Chewing gum was prepared according to the following formulation. Gum base 20.0% Sugar 53.0 Glucose 15.0 Minamata 10.5 Fragrance 0.5 Daisy extract of Example 1 1.0 100.0%
- Chewing gum was prepared according to the following formulation. Gum base 20.0% Sugar 54.0 Glucose 14.0 Minamata 9.3 Fragrance 0.7 Daisy extract of Example 1 2.0 100.0%
- Chocolate was prepared according to the following formulation. Cocoa bitter 20.0% Whole milk powder 20.0 Cocoa butter 17.0 Powdered sugar 41.7 Lecithin 0.5 Fragrance 0.2 Daisy extract of Example 1 0.6 100.0%
- Gummy jelly was prepared according to the following formulation. Polydextrose aqueous solution 38.0% Sorbitol aqueous solution 8.0 Palatinose aqueous solution 9.0 Maltose aqueous solution 20.0 Trehalose aqueous solution 11.0 Gelatin 10.0 Tartaric acid 1.0 Daisy extract of Example 1 3.0 100.0%
- Tablet confectionery was prepared according to the following formulation. 76.0% sugar Glucose 20.0 Sucrose fatty acid ester 0.2 Fragrance 0.1 Daisy extract of Example 1 0.5 Water 3.2 100.0%
- Tablet confectionery was prepared according to the following formulation.
- Daisy extract of Example 1 5.0 Water 3.0 100.0%
- Tablets were prepared according to the following formulation.
- Daisy extract of Example 1 70.0
- Ice cream was prepared according to the following formulation. Yolk 11.0% Sugar 13.5 Milk 37.0 Fresh cream 37.0 Vanilla beans 0.5 Daisy extract of Example 1 1.0 100.0%
- a sherbet was prepared according to the following formulation. Orange juice 20.0% 29.5 sugar Daisy extract of Example 1 0.5 Water 50.0 100.0%
- a beverage was prepared according to the following formulation. Orange juice 40.0% Isomerized sugar 15.3 Citric acid 0.1 Vitamin C 0.05 Fragrance 0.1 Daisy extract of Example 1 0.05 Water 44.4 100.0%
- a beverage was prepared according to the following formulation. Grape juice 70.0% Isomerized sugar 12.0 Citric acid 0.1 Vitamin C 0.05 Fragrance 0.1 Daisy extract of Example 1 0.1 Water appropriate amount 100.0%
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Abstract
Description
ヒナゲシ花100gを粉砕した後、試料の重量に対して10倍量の水を加えて70℃で2時間抽出した。不溶物を濾過した後、抽出液を凍結乾燥して熱水抽出物37.5gを得た。
マウス(BALB/c系、雌、6週齢)の小腸を摘出し、パイエル板を採取して2%ウシ胎児血清(FCS)を含むRPMI1620培地に浸した。ハサミでパイエル板を裁断し、セルストレーナー(BD Biosciences製、孔径70μm)でリンパ球細胞を回収した。回収した細胞懸濁液を遠心分離し、上清を吸引除去した後、complete mediumを加えてリンパ球細胞懸濁液の細胞濃度を4×106 cells/mlに調整した。complete mediumには10% FCS、50μM 2-メルカプトエタノール、1mMピルビン酸ナトリウム、100U/mlペニシリン、100μg/mlストレプトマイシンを含むRPMI1620培地を用いた。96穴プレートの各ウェルにリンパ球細胞懸濁液と試料を溶解した培養培地(試料は終濃度で100ppmとなるように調整)を0.05mlずつ加え、37℃、5%CO2の条件で1週間培養した。培養液を回収して遠心分離した後、上清中の総IgA抗体量をサンドイッチELISA法により測定した。試料を添加せずに培養した対照の総IgA抗体量を1とし、試料を添加して培養した場合の総IgA抗体量を相対値として算出した。
上記サンドイッチELISA法による測定は以下の手順により行った。
普通飼料(MF, オリエンタル酵母)にヒナゲシ(花)熱水抽出物を5%(w/w)混合した試験飼料を調製した。7週齢のマウス(BALB/c系)6匹を対照群と試験群(各群3匹)に分け、対照群には普通飼料、試験群には試験飼料を自由摂取させて6週間飼育した。飼育開始から1週間毎に糞便を回収し、それぞれ100mg/mlの濃度となるようにリン酸緩衝液に懸濁させ、遠心分離した後、上清中の総IgA抗体量を実施例2と同様にサンドイッチELISA法で測定した。
普通飼料(MF, オリエンタル酵母)にヒナゲシ(花)熱水抽出物を5%(w/w)混合した試験飼料を調製した。7週齢のマウス(BALB/c系)12匹を対照群と試験群(各群6匹)に分け、対照群には普通飼料、試験群には試験飼料を自由摂取させて飼育した。4週目に50μgのホスホリルコリン-keyhole limpet hemocyanin(PC-KLH、細菌に共通の抗原)を経鼻投与して免疫した。その後も1週間毎に経鼻免疫し、3回目の経鼻免疫から1週間後に鼻腔粘液(100~200μl/匹)及び鼻腔粘膜固有層リンパ球細胞(1×105~5×105cells/匹)を回収し、総IgA抗体量及び総IgA抗体産生細胞数、PC特異的IgA抗体価及びPC特異的IgA抗体産生細胞数を測定した。また、糞便を回収し、100mg/mlの濃度となるようにリン酸緩衝液に懸濁させ、遠心分離した後、上清中の総IgA抗体量を測定した。総IgA抗体量及びPC特異的IgA抗体価の測定はサンドイッチELISA法で、PC特異的IgA抗体産生細胞数の測定はenzyme-linked immunospot(ELISPOT)法で行った。尚、各動物から採取できる鼻腔粘膜固有層リンパ球細胞の数は非常に少ないため、3匹分の細胞を一つにまとめて2連でIgA抗体産生細胞数の測定を行った。
上記総IgA抗体量の測定は実施例2と同様にサンドイッチELISA法により行った。また、上記PC特異的IgA抗体量の測定は、最初にELISAプレートに吸着させる抗マウスイムノグロブリン抗体の代わりにPC-BSAを用いたほかは実施例2と同様にしてサンドイッチELISA法により行った。
上記ELISPOT法によるPC特異的IgA抗体産生細胞数測定は以下の手順により行った。
CO2の条件下で4時間インキュベートした。PBSで洗浄後、100ngのペルオキシダーゼ結合抗マウスIgA抗体を加え、4℃で一晩反応させた。PBSで洗浄後、発色基質(3-amino-9-ethylcarbazole)液を加えて室温、遮光条件で30分発色させた。水で洗浄し乾燥させた後に、spotを抗体産生細胞として実体顕微鏡下にて計測した。
糞便を回収し、100mg/mlの濃度となるようにPBSに懸濁させ、遠心分離した後、上清の総IgA量を実施例2と同様にサンドイッチELISA法で測定した。
普通飼料(MF, オリエンタル酵母)にヒナゲシ(花)熱水抽出物を5%(w/w)混合した試験飼料を調製した。7週齢のマウス(BALB/c系)20匹を対照群と試験群(各群10匹)に分け、実施例4と同様の方法で鼻腔粘液の総IgA抗体量及びPC特異的IgA抗体価、糞便の総IgA抗体量を測定した。
7週齢のマウス(BALB/c系)を1群4匹として対照群と投与群に分け、普通飼料(MF, オリエンタル酵母)で飼育した。投与群には飼育開始時からヒナゲシ(花)熱水抽出物を4mg/dayの用量で水溶液として毎日舌下投与し、対照群は同量の水のみを舌下投与した。4週目に50μgのPC-KLHを経鼻投与して免疫し、その後も1週間毎に経鼻免疫して、3回目の経鼻免疫から1週間後に鼻腔粘液及び鼻腔粘膜固有層リンパ球細胞を回収し、総IgA抗体量及び総IgA抗体産生細胞数を測定した。総IgA抗体量の測定は実施例2と同様にサンドイッチELISA法で、IgA抗体産生細胞数の測定は実施例4と同様にenzyme-linked immunospot(ELISPOT)法で行った。尚、各動物から採取できる鼻腔粘膜固有層リンパ球細胞の数は非常に少ないので、4匹分の細胞をまとめて、2連でIgA抗体産生細胞数の測定を行った。
7週齢のマウス(BALB/c系)を1群15匹として対照群と投与群に分け、普通飼料(MF, オリエンタル酵母)で飼育した。投与群には飼育開始時からヒナゲシ(花)熱水抽出物を4mg/dayの用量で水溶液として毎日舌下投与し、対照群は同量の水のみを舌下投与した。4週目に50μgのPC-KLHを経鼻投与して免疫し、その後も1週間毎に経鼻免疫して、3回目の経鼻免疫から1週間後に鼻腔粘液及び鼻腔粘膜固有層リンパ球細胞を回収し、総IgA抗体量及び総IgA抗体産生細胞数、PC特異的IgA抗体価及びPC特異的IgA抗体産生細胞数を測定した。また、糞便を回収してリン酸緩衝液に懸濁させ、遠心分離した後、上清中の総IgA抗体量を測定した。総IgA抗体量及びPC特異的IgA抗体価の測定は実施例2と同様にサンドイッチELISA法で、IgA抗体産生細胞数の測定は実施例4と同様にenzyme-linked immunospot(ELISPOT)法で行った。尚、各動物から採取できる鼻腔粘膜固有層リンパ球細胞の数は非常に少ないので、3匹分の細胞をまとめて、2連でIgA抗体産生細胞数の測定を行った。
7週齢のマウス(BALB/c系)を1群6匹として対照群と投与群に分け、普通飼料(MF, オリエンタル酵母)で飼育した。投与群には飼育開始時からヒナゲシ(花)熱水抽出物を2mg/dayの用量で水溶液として毎日経鼻投与し、対照群は同量の水のみを経鼻投与した。4週目に50μgのPC-KLHを経鼻投与して免疫し、その後も1週間毎に経鼻免疫して、3回目の経鼻免疫から1週間後に鼻腔粘液及び鼻腔粘膜固有層リンパ球細胞を回収し、総IgA抗体量及び総IgA抗体産生細胞数を測定した。総IgA抗体量の測定は実施例2と同様にサンドイッチELISA法で、IgA抗体産生細胞数の測定は実施例4と同様にenzyme-linked immunospot(ELISPOT)法で行った。尚、各動物から採取できる鼻腔粘膜固有層リンパ球細胞の数は非常に少ないので、3匹分の細胞をまとめて、2連でIgA抗体産生細胞数の測定を行った。
5~6ヶ月齢のマウス(BALB/c系)を1群5匹として対照群と投与群に分け、普通飼料(MF, オリエンタル酵母)で飼育した。投与群には飼育開始時からヒナゲシ(花)熱水抽出物を2mg/dayの用量で水溶液として毎日経鼻投与し、対照群は同量の水のみを経鼻投与した。4週目に50μgのPC-KLHを経鼻投与して免疫し、その後も1週間毎に経鼻免疫して、3回目の経鼻免疫から1週間後に鼻腔粘液及び鼻腔粘膜固有層リンパ球細胞を回収し、総IgA抗体量及び総IgA抗体産生細胞数を測定した。総IgA抗体量の測定は実施例2と同様にサンドイッチELISA法で、IgA抗体産生細胞数の測定は実施例4と同様にenzyme-linked immunospot(ELISPOT)法で行った。尚、各動物から採取できる鼻腔粘膜固有層リンパ球細胞の数は非常に少ないので、2~3匹分の細胞をまとめて、2連でIgA抗体産生細胞数の測定を行った。
D-マンニトール 35.0%
乳糖 27.5
結晶セルロース 12.5
ヒドロキシプロピルセルロース 5.0
実施例1のヒナゲシ抽出物 20.0
100.0%
乳糖 54.5%
トウモロコシデンプン 38.5
ステアリン酸マグネシウム 2.0
実施例1のヒナゲシ抽出物 5.0
100.0%
乳糖 8.0%
ステアリン酸マグネシウム 2.0
実施例1のヒナゲシ抽出物 90.0
100.0%
乳糖 40.0%
馬鈴薯でんぷん 15.0
実施例1のヒナゲシ抽出物 45.0
100.0%
エタノール 5.0%
実施例1のヒナゲシ抽出物 0.5
L-メントール 1.0
水 93.5
100.0%
サリチル酸メチル 0.03g
マレイン酸クロルフェニラミン 0.3
dl―塩酸メチルエフェドリン 0.3
ポリソルベート80 0.2
塩化ベンザルコニウム 0.01
塩化ナトリウム 0.6
1N水酸化ナトリウム 適量
実施例1のヒナゲシ抽出物 0.01
水 適量
100.0ml
(pH6.8)
ガムベース 20.0%
砂糖 52.0
グルコース 14.0
水飴 13.0
香料 0.5
実施例1のヒナゲシ抽出物 0.5
100.0%
ガムベース 20.0%
砂糖 53.0
グルコース 15.0
水飴 10.5
香料 0.5
実施例1のヒナゲシ抽出物 1.0
100.0%
ガムベース 20.0%
砂糖 54.0
グルコース 14.0
水飴 9.3
香料 0.7
実施例1のヒナゲシ抽出物 2.0
100.0%
砂糖 45.0%
水飴 40.0
香料 0.5
L-メントール 0.3
実施例1のヒナゲシ抽出物 0.2
水 14.0
100.0%
砂糖 50.0%
水飴 33.0
クエン酸 1.0
香料 0.6
実施例1のヒナゲシ抽出物 1.0
水 14.4
100.0%
カカオビター 20.0%
全脂粉乳 20.0
カカオバター 17.0
粉糖 41.7
レシチン 0.5
香料 0.2
実施例1のヒナゲシ抽出物 0.6
100.0%
砂糖 31.0%
小麦粉 26.5
片栗粉 26.5
バター 4.0
卵 10.5
重曹 0.3
実施例1のヒナゲシ抽出物 1.2
100.0%
ポリデキストロース水溶液 38.0%
ソルビトール水溶液 8.0
パラチノース水溶液 9.0
マルトース水溶液 20.0
トレハロース水溶液 11.0
ゼラチン 10.0
酒石酸 1.0
実施例1のヒナゲシ抽出物 3.0
100.0%
砂糖 76.0%
グルコース 20.0
ショ糖脂肪酸エステル 0.2
香料 0.1
実施例1のヒナゲシ抽出物 0.5
水 3.2
100.0%
砂糖 72.0%
乳糖 19.8
ショ糖脂肪酸エステル 0.2
実施例1のヒナゲシ抽出物 5.0
水 3.0
100.0%
砂糖 28.8%
ショ糖脂肪酸エステル 0.2
実施例1のヒナゲシ抽出物 70.0
水 1.0
100.0%
卵黄 11.0%
砂糖 13.5
牛乳 37.0
生クリーム 37.0
バニラビーンズ 0.5
実施例1のヒナゲシ抽出物 1.0
100.0%
オレンジ果汁 20.0%
砂糖 29.5
実施例1のヒナゲシ抽出物 0.5
水 50.0
100.0%
オレンジ果汁 40.0%
異性化糖 15.3
クエン酸 0.1
ビタミンC 0.05
香料 0.1
実施例1のヒナゲシ抽出物 0.05
水 44.4
100.0%
グレープ果汁 70.0%
異性化糖 12.0
クエン酸 0.1
ビタミンC 0.05
香料 0.1
実施例1のヒナゲシ抽出物 0.1
水 適量
100.0%
Claims (7)
- ヒナゲシ抽出物を含有することを特徴とする、IgA分泌促進剤。
- 請求項1に記載のIgA分泌促進剤を乾燥重量に換算して0.01%~90%含有することを特徴とする医薬組成物。
- 経口摂取されることを特徴とする請求項2に記載の医薬組成物。
- 舌下投与により摂取されることを特徴とする請求項2に記載の医薬組成物。
- 経鼻投与により摂取されることを特徴とする請求項2に記載の医薬組成物。
- 請求項1に記載のIgA分泌促進剤を含有することを特徴とする食品。
- 請求項1に記載のIgA分泌促進剤を含有することを特徴とする医薬組成物。
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JP2012503020A JP5879254B2 (ja) | 2010-03-04 | 2011-03-03 | イムノグロブリンa分泌促進剤 |
BR112012022203A BR112012022203A2 (pt) | 2010-03-04 | 2011-03-03 | um promotor de secreção de imunoglobulina |
EP11750388.8A EP2543381A4 (en) | 2010-03-04 | 2011-03-03 | IMMUNE LOBULIN AS A SEPARATION CONVEYOR |
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JP7313519B1 (ja) * | 2022-07-08 | 2023-07-24 | 日本甜菜製糖株式会社 | IgA産生促進剤及びIgA産生促進用の飼料組成物 |
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- 2011-03-03 CN CN201180012014.XA patent/CN102781457B/zh not_active Expired - Fee Related
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- 2011-03-03 JP JP2012503020A patent/JP5879254B2/ja active Active
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Also Published As
Publication number | Publication date |
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EP2543381A1 (en) | 2013-01-09 |
JPWO2011108275A1 (ja) | 2013-06-20 |
HK1176546A1 (en) | 2013-08-02 |
EP2543381A4 (en) | 2013-09-18 |
KR20130037672A (ko) | 2013-04-16 |
JP5879254B2 (ja) | 2016-03-08 |
BR112012022203A2 (pt) | 2016-07-05 |
CN102781457A (zh) | 2012-11-14 |
KR101896123B1 (ko) | 2018-09-07 |
TW201141499A (en) | 2011-12-01 |
CN102781457B (zh) | 2015-01-14 |
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