WO2016190395A1 - ペプチドを含む炎症抑制のための組成物 - Google Patents
ペプチドを含む炎症抑制のための組成物 Download PDFInfo
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- WO2016190395A1 WO2016190395A1 PCT/JP2016/065627 JP2016065627W WO2016190395A1 WO 2016190395 A1 WO2016190395 A1 WO 2016190395A1 JP 2016065627 W JP2016065627 W JP 2016065627W WO 2016190395 A1 WO2016190395 A1 WO 2016190395A1
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- 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
- A23L33/17—Amino acids, peptides or proteins
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- 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
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention relates to a composition for suppressing inflammation of microglia and its use for food and drink.
- Microglia which exists as the only immune cell in the brain, occupies 10% of the brain and has essential functions for maintaining homeostasis in the brain, such as phagocytosis of waste products and repair of damaged tissues. It is clear to have. Microglia contribute to the maintenance, enhancement, and improvement of cognitive function by maintaining homeostasis in the brain. However, it is known that inflammation is caused by excessive activation of microglia, and inflammatory cytokines such as reactive oxygen (ROS), TNF- ⁇ , and IL-1 ⁇ are chronically produced and stress neurons. .
- ROS reactive oxygen
- TNF- ⁇ TNF- ⁇
- IL-1 ⁇ chronically produced and stress neurons.
- Non-Patent Document 1 Non-Patent Document 1
- Inflammatory cytokines such as reactive oxygen and TNF- ⁇ produced by over-activated microglia are reported to be closely related to pain and chronic fatigue syndrome in addition to mood disorders including depression (Non-Patent Documents 2 to 7). From these findings, if it is possible to suppress excessive activation of microglia, diseases that are correlated with excessive activation of microglia, such as depression, pain, chronic fatigue syndrome, dementia, and multiple sclerosis It is thought to be useful in the treatment, alleviation, and prevention.
- Patent Document 1 discloses a peptide that suppresses inflammation of microglia and protects nerves. It has been shown that a polypeptide comprising a peptide consisting of 5 amino acids having specific properties in its sequence has an action of suppressing the activation of microglia and suppressing inflammation. In addition, it has been suggested that the peptide may be effective for various diseases including acute diseases and chronic diseases that are considered to be related to inflammation of microglia. Further, Patent Document 1 describes administration by injection, inhalation, etc. as a peptide administration method.
- Patent Document 2 discloses that nerves are continuously protected by intravenous administration of the tripeptide Gly-Pro-Glu (GPE). In particular, it is intended for diseases with morphologically significant damage such as acute ischemic damage. Moreover, it is not disclosed that the target is microglia.
- GPE tripeptide Gly-Pro-Glu
- Patent Documents 1 and 2 are premised on administration by injection for diseases associated with neurodegeneration. Therefore, it is understood that the aim was to administer to patients with relatively severe symptoms.
- compositions that are effective not only for patients with severe symptoms but also for those with relatively mild symptoms and that can be ingested continuously.
- An object of the composition of the present invention is to provide a composition that is effective not only for patients developing these diseases, but also for groups with a high risk of onset.
- Inflammation in the brain caused by chronic stress in daily life is maintained in a normal state by suppressing excessive inflammation of microglia.
- stress resistant normal state it is considered important that the inflammation of microglia is appropriately controlled.
- An object of the present invention is to provide a peptide having an effect on various diseases and conditions caused by inflammation of microglia, or a composition containing a pharmaceutically acceptable salt or solvate thereof as an active ingredient.
- Compositions and foods and drinks that alleviate and suppress the inflammatory conditions caused by microglia in the brain and improve not only diseases such as chronic fatigue syndrome, dementia, and mood disorders, but also the conditions observed before onset of the disease
- the issue is to provide goods.
- the first aspect of the present invention includes a dipeptide having an amino acid sequence represented by LH, DV or MH, or an oligopeptide containing the amino acid sequence as a core sequence, or a pharmaceutically acceptable salt or solvate thereof.
- the present invention provides a composition for suppressing inflammation of microglia.
- a dipeptide having an amino acid sequence represented by LH, DV or MH, or an oligopeptide containing the amino acid sequence as a core sequence has an action of suppressing inflammation of microglia. Therefore, the composition containing this can be expected to have an effect of suppressing inflammation of microglia.
- the second aspect of the present invention includes a dipeptide having an amino acid sequence represented by LH, DV or MH, or an oligopeptide containing the amino acid sequence as a core sequence, or a pharmaceutically acceptable salt or solvate thereof.
- the present invention provides a composition for alleviating, treating, or preventing symptoms of chronic fatigue syndrome, dementia and / or mood disorders.
- a dipeptide having an amino acid sequence represented by LH, DV or MH, or an oligopeptide containing the amino acid sequence as a core sequence has an action of suppressing inflammation of microglia.
- Microglial inflammation has been associated with chronic fatigue syndrome, dementia, and mood disorders such as depression. Therefore, a composition containing the above-mentioned dipeptide or a composition containing an oligopeptide containing the amino acid sequence as a core sequence suppresses the inflammatory action of microglia, thereby preventing chronic fatigue syndrome, dementia and / or mood disorder. Expected to relieve, treat, or prevent symptoms.
- the third aspect of the present invention includes a dipeptide having an amino acid sequence represented by LH, DV or MH, or an oligopeptide containing the amino acid sequence as a core sequence, or a pharmaceutically acceptable salt or solvate thereof.
- the present invention provides a composition for alleviating or preventing a state caused by stress.
- a dipeptide having an amino acid sequence represented by LH, DV or MH, or an oligopeptide containing the amino acid sequence as a core sequence has an action of suppressing inflammation of microglia.
- Microglial inflammation has been associated with stress-induced conditions. Therefore, a composition containing the above-mentioned dipeptide or a composition containing an oligopeptide containing the amino acid sequence as a core sequence is expected to have an effect of alleviating or preventing a state caused by stress by suppressing the inflammatory action of microglia. it can.
- Conditions caused by stress include, among other things, lack of motivation and motivation and / or reduced vitality.
- the composition may be a food composition.
- the composition may be a pharmaceutical composition.
- the composition may be contained in a food or drink.
- the composition is a dipeptide having an amino acid sequence represented by LH, DV or MH in each composition, an oligopeptide containing the amino acid sequence as a core sequence, or a pharmaceutically acceptable salt or solvent thereof. It can be produced by incorporating a Japanese product.
- the above-mentioned dipeptide or oligopeptide containing the amino acid sequence as a core sequence may be obtained by hydrolyzing a protein derived from food or the like. In particular, it may be contained in sake lees. In this case, the protein hydrolyzate or the sake lees may be further purified and concentrated to increase the concentration of the dipeptide or the oligopeptide containing the amino acid sequence as a core sequence in the composition.
- dipeptide or oligopeptide containing the amino acid sequence as a core sequence is derived from food or the like, it is possible to provide a composition that can be consumed with peace of mind.
- the first form of the production method of the present invention is a production method of the above composition, wherein a dipeptide having an amino acid sequence represented by LH, DV or MH, or the amino acid sequence is hydrolyzed.
- a method for producing a composition comprising a step of obtaining an oligopeptide comprising a core sequence, or a pharmaceutically acceptable salt or solvate thereof.
- the protein is hydrolyzed to obtain a dipeptide having an amino acid sequence represented by LH, DV or MH, or an oligopeptide containing the amino acid sequence as a core sequence, or a pharmaceutically acceptable salt or solvate thereof.
- the protein is hydrolyzed to obtain a dipeptide having an amino acid sequence represented by LH, DV or MH, or an oligopeptide containing the amino acid sequence as a core sequence, or a pharmaceutically acceptable salt or solvate thereof.
- the composition obtained by the process may be a sake lees.
- a second aspect of the production method of the present invention is a method for producing a composition for alleviating or preventing a state caused by stress, comprising a dipeptide having an amino acid sequence represented by LH, DV, or MH, or Provided is a production method including a step of containing an oligopeptide containing an amino acid sequence as a core sequence, or a sake cake containing a pharmaceutically acceptable salt or solvate thereof.
- a composition having a function of suppressing inflammation of microglia can be provided. And by taking it, not only relief, treatment, prevention and alleviation of symptoms such as chronic fatigue syndrome, dementia, mood disorders caused by microglial inflammation, but also diseases caused by stress It is possible to alleviate, prevent, and improve conditions that are not diagnosed.
- Experiment 2 it is a figure which shows the result of having investigated the inflammation inhibitory effect with respect to microglia by a dipeptide.
- Experiment 3 it is a figure which shows the result of having investigated the inflammation inhibitory effect with respect to the microglia by the tripeptide and tetrapeptide which have a dipeptide as a core sequence.
- 10 is a chart showing an outline of a test schedule in Test Example 4.
- Experiment 4 it is a figure which shows the result of having investigated the inflammation-inhibition effect in the brain by administration of the dipeptide in the mouse brain. It is a test conceptual diagram of the social interaction test in Test Example 5.
- 10 is a chart showing an outline of a test schedule in Test Example 5.
- FIG. 5 It is a figure which shows the outline
- FIG. It is a figure which shows the result of having investigated the time-dependent change of Avoidance
- a dipeptide having an amino acid sequence represented by LH, DV or MH, or an oligopeptide containing the amino acid sequence as a core sequence is used in a composition for suppressing inflammation of microglia.
- the oligopeptide specifically refers to a tripeptide or tetrapeptide containing the above-mentioned dipeptide as a core sequence. You may use these in combination of multiple types. Hereinafter, these may be simply referred to as “dipeptide” or “oligopeptide”.
- the amino acid constituting the dipeptide or oligopeptide used in the present invention may be composed only of the L-form amino acid, may be composed only of the D-form amino acid, or a dipeptide or oligo in which both are mixed. Any of peptides may be sufficient. Further, it may be composed only of naturally occurring amino acids, may be composed only of modified amino acids in which any functional group is bonded to amino acids, or may be a dipeptide or oligopeptide in which both are mixed. There may be. When the dipeptide or oligopeptide contains two or more asymmetric carbons, it may be an enantiomer, a diastereomer, or a dipeptide or oligopeptide in which both are mixed.
- the dipeptide or oligopeptide used in the present invention may be a pharmaceutically acceptable salt or solvate thereof.
- pharmaceutically acceptable acid addition salts include inorganic acid salts such as hydrochloride, sulfate and phosphate, organic salts such as acetate, maleate, fumarate, citrate and methanesulfonate. Examples include acid salts.
- pharmaceutically acceptable metal salt include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, aluminum salt and zinc salt.
- Examples of the pharmaceutically acceptable ammonium salt include salts such as ammonium and tetramethylammonium.
- pharmaceutically acceptable organic amine addition salts include addition salts of morpholine, piperidine and the like.
- Dipeptides or oligopeptides used in the present invention may be those obtained by chemical synthesis, such as proteins derived from milk, soybeans, wheat, eggs, livestock, fish, seafood, barley, rice, sweet potatoes, potatoes, etc. Those obtained by chemically or enzymatically decomposing from a polypeptide raw material or the like may be used. Specifically, for example, any of the sequences of the dipeptide or oligopeptide used in the present invention is at least alpha protein casein, beta casein, kappa casein, lactoglobulin, lactalbumin, immunoglobulin, lactoperoxidase, lactoferrin, albumin.
- a composition containing about 1% of the above-mentioned dipeptide or oligopeptide can be obtained by dissolving skim milk powder or defatted soybean protein and appropriately treating with enzyme.
- the decomposition includes fermentation, and as a food composition obtained by fermenting a raw material containing protein, alcoholic beverages, liquors, miso, miso, yogurt, cheese, fermented milk Natto, vinegar, potatoes, soy sauce (including fish sauce), fermented fish such as salted and anchovies, fermented meat such as salami, and aged meat.
- the composition may be used as it is, or may be used after further purification and concentration of any degree.
- Purification / concentration means increasing the content of the dipeptide or oligopeptide in the composition by means of, for example, separation, fractionation, extraction, dialysis, salting out, reprecipitation, membrane treatment, etc. May be applied in combination.
- extraction more preferably water extraction
- membrane treatment is selected.
- dipeptide or oligopeptide used in the present invention can be analyzed and quantified by an appropriate method as appropriate by those skilled in the art.
- dipeptides or oligopeptides in compositions and foods can be analyzed and quantified by LC / MSMS shown in Examples below.
- Mood disorders are defined according to the World Health Organization (WHO) international statistical classification of diseases and related health problems as conditions that interfere with daily life due to mood modulation that persists over a period of time. ing.
- mood disorders include even milder symptoms such as a state of reduced vitality and a state of curiosity.
- the improvement of vitality and the improvement of curiosity in the present invention include the following states.
- the improvement of vitality means, for example, that the amount of physical activity is greater after ingestion than before the intake of the composition of the present invention, and the improvement of curiosity means that, for example, an interest in a new problem is shown.
- Stress is a pressure in life and a sense when it is felt.
- Enhance vitality and curiosity can be measured using experimental animals such as mice as follows.
- the improvement of vitality can be evaluated by comparing the operation time of a pulley such as a rotor rod in a certain time before and after taking the composition in a mouse model.
- the improvement of curiosity can be achieved by, for example, giving a new toy to the mouse model before and after taking the composition and comparing the time to start operating the toy or the time to operate the toy. It can be evaluated by comparing sociality such as how much approach to the mouse when encountering another new mouse.
- the composition of the present invention can be used as a food composition, an additive used for blending in the food composition, a pharmaceutical composition, an additive used for blending in the pharmaceutical composition, or the like.
- it may be used not only for humans but also for pets (dogs, cats, reptiles, birds, fishes, etc.), livestock (including poultry), farmed fish, etc. (preferably used for animals and livestock that are mammals).
- the dipeptide or oligopeptide (when multiple types are included, the total amount) should be in a form containing 0.00001 to 100% by mass.
- a form containing 0.0001 to 100% by mass is more preferable, and a form containing 0.001 to 100% by mass is most preferable.
- the above-mentioned dipeptide or oligopeptide can be used in a method for suppressing inflammation of microglia. Moreover, it can be used for the method of relieving, treating, or preventing the symptoms of chronic fatigue syndrome, dementia and / or mood disorder. Moreover, it can be used for the method of relieving or preventing the state caused by stress, such as lack of motivation and motivation, and / or decline of vitality, for example. They can be performed by ingesting or administering an effective amount of the above dipeptide or oligopeptide. The method does not include so-called medical practice in humans.
- composition provided by the present invention is not particularly limited in its form, and may be liquid, semi-liquid, or solid, and includes food composition forms such as beverages.
- health foods specific insurance foods, nutritional functional foods, functional labeling foods), diseases, etc., including so-called health foods, functional foods, dietary supplements, supplements, and foods with a disease risk reduction label. Consumer food is also included.
- the protein in the raw material is degraded by the added enzyme / microorganism, or as a result of degradation by the enzyme originally contained in the raw material, Oligopeptides are included.
- the raw materials for vinegars and liquors may include proteins, and preferably include wheat (including malt), rice (including germinated rice), potatoes, corn, beans, and buckwheat. More preferred are barley, rice, and sweet potato.
- liquor squeezing is more preferable.
- the wringer may or may not contain alcohol, but preferably does not contain alcohol.
- the alcohol may be removed by a known method such as air drying.
- alcoholic beverages are mainly produced by (i) primary treatment of raw materials (such as whiskey and fruit wine) (decomposition treatment by addition of enzymes / microorganisms, or decomposition treatment by enzymes derived from the raw materials themselves) and solid-liquid separation.
- raw materials such as whiskey and fruit wine
- solid-liquid separation A method of obtaining liquor by subjecting the liquid to a fermentation process later, and (ii) primary treatment of the raw material such as sake or shochu (addition of enzymes / microorganisms or decomposition treatment with an enzyme derived from the raw material itself)
- there is a method in which raw materials, enzymes and microorganisms are added to perform a fermentation process, and after fermentation, solid-liquid separation and distillation are performed to obtain sake.
- the liquor of the liquor of the present invention may be obtained by any of (i) and (ii), but is preferably obtained by (ii).
- Examples of the solid obtained in (i) include whiskey koji and grape koji, and examples of the solid obtained in (ii) include sake lees and shochu.
- the squeezed lees of liquors are collectively referred to as sake lees.
- Non-alcoholic beverages when the food composition is applied to non-alcoholic beverages include, for example, mineral water, near water, sports drinks, tea beverages, milk beverages, coffee beverages, fruit juice beverages, vegetable juice beverages, fruit juices and Examples include vegetable juice drinks, carbonated drinks, and beer-taste drinks that do not contain alcohol, but are not limited thereto. It may be a beer beverage having an alcohol content of less than 1%, such as non-alcohol beer.
- Mineral water includes both foaming and non-foaming mineral water.
- the tea drink in the above non-alcoholic drinks refers to drinks made from leaves of tea trees (tea leaves) that are evergreen trees of the camellia family, or leaves or grains of plants other than tea trees, such as fermented tea, semi-fermented tea, And non-fermented tea are both included.
- tea beverages include Japanese tea (for example, green tea, barley tea), black tea, herbal tea (for example, jasmine tea), Chinese tea (for example, Chinese green tea, oolong tea), hoji tea, and the like.
- Milk beverages refer to beverages made mainly from raw milk, milk, etc. or foods made from these raw materials. In addition to those made from milk itself, for example, nutrient-enriched milk, flavored milk, and sugar-decomposed milk And the like using processed milk as a raw material.
- fruits used in beverages containing fruit juices and beverages containing fruit juices and vegetable juices include apples, mandarin oranges, grapes, bananas, pears, peaches, mangoes, acais, and blueberries.
- vegetables used in beverages containing vegetable juice, fruit juices, and beverages containing vegetable juice include tomatoes, carrots, celery, pumpkins, and cucumbers.
- the daily intake per day for adults and adults is 0.0001 to 40 g of the above-mentioned dipeptide or oligopeptide, preferably 0.001 to 20 g of the above-mentioned dipeptide or oligopeptide, and even more preferably the above-mentioned dipeptide or oligopeptide. 0.001 to 2 g of oligopeptide (when multiple types are included, the total is converted).
- the dipeptide or oligopeptide (in the case of including a plurality of types) is preferably in a form containing 0.00001 to 100% by mass, 0.0001 More preferred is a form containing from 100 to 100% by weight, still more preferred is a form containing from 0.001 to 100% by weight, still more preferred is a form containing from 0.01 to 90% by weight, The most preferred form is 1 to 80% by mass.
- 0.0001 to 40 g of the above-mentioned dipeptide or oligopeptide (when converted into a total amount) is contained in a container subdivided into one serving or one serving, and 0.001 to A form containing 20 g is more preferable, and a form containing 0.001 to 2 g is even more preferable.
- the above-mentioned dipeptide or oligopeptide has an anti-inflammatory action / stress mitigating action of microglia, it can be provided even if it is contained in foods taken daily or foods taken as supplements. It can also be added to health foods and functional foods, preferably foods containing ingredients intended to exert microglial inflammation-inhibiting action, mood disorder-inhibiting action, or stress-relaxing effect.
- Ingredients intended to exert microglial inflammation-inhibiting, mood disorder-inhibiting, or stress-reducing effects include, for example, theanine, polyphenols, rosemary hydrolysates, ubidecalenone, sorghum, actin, lactic acid bacteria fermented soy milk, ⁇ Such as a mall.
- the treatment of long-term mood disorders can prevent the transition to cognitive dysfunction to some extent, so when taking food intended for the prevention and treatment of cognitive dysfunction, In contrast, it may be useful to ingest ingredients that can alleviate or improve the state of reduced resistance. Since the composition provided by the present invention has a microglial inflammation inhibitory action, it can be further contained in a food containing components intended for the prevention / treatment of cognitive impairment.
- ingredients intended to prevent cognitive impairment include omega-3 fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), polyphenols such as ginkgo biloba extract, resveratrol, curcumin, lecithin, isohumulone Vitamins that prevent homocysteine metabolism abnormalities, a risk factor for Alzheimer's disease.
- omega-3 fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)
- polyphenols such as ginkgo biloba extract, resveratrol, curcumin, lecithin
- isohumulone Vitamins that prevent homocysteine metabolism abnormalities, a risk factor for Alzheimer's disease.
- the above-mentioned dipeptide or oligopeptide is effective even after a single ingestion, but by taking it continuously as a food or drink, the excessive inflammatory action of microglia can be suppressed and adjusted appropriately. It is also possible to reduce the vulnerability to the stress, that is, to provide a resistance to stress.
- composition can be produced by mixing at least one of the above-mentioned dipeptides or oligopeptides as an active ingredient and mixing a carrier, excipient, binder, diluent and the like. It can be administered orally or parenterally, and when administered orally, it may be in any form such as granules, powders, tablets, pills, capsules, syrups and the like. Examples of parenteral dosage forms include, but are not limited to, external preparations such as injections, drops, and nasal preparations.
- the dosage When used as a pharmaceutical, the dosage varies depending on the dosage form, patient age, body weight, nature or severity of the condition to be treated, etc., but the oral dosage is usually per adult per day, 0.0001 to 40 g of the above-mentioned dipeptide or oligopeptide, preferably 0.001 to 20 g of the above-mentioned dipeptide or oligopeptide, even more preferably 0.001 to 2 g of the above-mentioned dipeptide or oligopeptide. Total conversion).
- the dose for intravenous administration is 0.00001 to 4 g of the above-mentioned dipeptide or oligopeptide, preferably 0.0001 to 2 g of the above-mentioned dipeptide or oligopeptide, more preferably still. Is 0.0001 to 0.2 g of the above-mentioned dipeptide or oligopeptide (when plural types are included, the total is converted).
- an optimal form may be appropriately selected according to various conditions.
- the above-mentioned dipeptide or oligopeptide has a relatively small molecular weight, after ingestion into the body, it easily passes through the brain barrier and easily exerts its effect in the brain.
- a protein contained in food is used as a raw material, a highly safe composition can be provided.
- Microglia were isolated from mouse brain by magnetic cell separation. Specifically, brain tissue dispersion was obtained by papain treatment of the brain removed from the mouse, and the enzyme reaction was stopped. Next, by reacting with a CD11b antibody (manufactured by Miltenyi Biotec), which is a pan-microglia marker magnetically labeled with superparamagnetic microbeads, and magnetically separating it, Microglia were isolated.
- a CD11b antibody manufactured by Miltenyi Biotec
- the isolated microglia cells were cultured for 12 hours in a medium supplemented with the above 336 dipeptides at a concentration of 50 ⁇ M. Thereafter, 5 ng / ml lipopolyshccaride (LPS, manufactured by SIGMA-ALDRICH) and 0.5 ng / ml IFN- ⁇ (manufactured by R & D system) were added, followed by further culturing for 12 hours, and TNF- contained in the culture supernatant. ⁇ was quantified using an ELISA kit (manufactured by eBioscience). Wells to which no peptide was added were used as controls, and the production ratio of TNF- ⁇ when the control was 1 was calculated. The results are shown in Table 1. The result is an average value of triplicate data.
- amino acids are indicated by single letter abbreviations.
- dipeptides having an activity of suppressing TNF- ⁇ production to 0.80 or less with respect to controls are AG, AH, AI, DA, DD, DF, DG, DH, DT, DV , DY, EA, EG, EM, EN, EW, EY, FA, FD, FI, FL, FM, FW, FY, GH, GS, GT, HS, HV, IE, IF, IQ, IR, IS, KD , KE, KG, KI, KK, KL, KM, KN, KQ, KR, KT, KV, KW, KY, LA, LD, LF, LG, LH, LI, LL, LV, LY, MA, MD, ME , MF, MH, MM, MN, MQ, MS, MT, MV, MW, MY, NA, ND, NE, NF, NG, NH, NI, NK,
- dipeptides having the activity of suppressing the production of TNF- ⁇ by a quarter are AG, AH, AI, DA, DD, DH, DV, EA, EY. , GH, IE, IQ, KI, KL, KM, KQ, KT, KV, KY, LG, LH, LI, LV, LY, MA, MD, ME, MF, MH, MN, MT, MW, MY, NA ND, NF, NG, NH, NL, NM, PS, PW, QF, QH, QI, QK, QQ, RF, RK, SL.
- dipeptides having an activity of suppressing TNF- ⁇ production to 0.70 or less are AI, DV, GH, KI, KL, KM, KV, LG, LH, LI, MA, MD, MH, NA.
- dipeptides having an activity of suppressing TNF- ⁇ production to 0.70 or less are AI, DV, GH, KI, KL, KM, KV, LG, LH, LI, MA, MD, MH, NA.
- the three dipeptides, LH, DV, and MH which suppress to 0.5 or less, have very excellent inflammation suppressing effects of TNF- ⁇ production of 0.06, 0.20, and 0.44, respectively. I found out.
- TNF- ⁇ The production of TNF- ⁇ was measured by ELISA in the same manner as in Test Example 1 except that the concentration of dipeptide was varied from 1 ⁇ M to 50 ⁇ M and cultured with microglia. In addition, as a control, the same as in Test Example 1, which was cultured without adding a peptide was used. For comparison, the production of TNF- ⁇ was also measured by changing the concentration of dipeptides having the same amino acid composition and different sequences, ie, VD and HL. The results are shown in FIG.
- dipeptides DV and LH As shown in FIG. 1, the anti-inflammatory action of dipeptides DV and LH is concentration-dependent.
- the dipeptides VD and HL having a sequence in which the N terminus and the C terminus were exchanged did not show anti-inflammatory activity.
- TNF- ⁇ was measured by ELISA in the same manner as in Test Example 1, except that microglia were cultured using the above peptides at concentrations of 10 ⁇ M and 50 ⁇ M, respectively. The results are shown in FIG.
- oligopeptides used in the analysis effects other than DVE were found to suppress the inflammatory action of microglia.
- effects other than DVE were found to suppress the inflammatory action of microglia.
- TDVE has the strongest anti-inflammatory effect, and the presence or absence of T does not affect the inflammatory action.
- the dipeptide sequence disclosed in the present invention is provided as the core sequence, even if it is used as a tripeptide or tetrapeptide, it may have an anti-inflammatory effect. There is sex.
- LPS manufactured by SIGMA-ALDRICH
- distilled water for the purpose of inducing inflammation in the brain, LPS (manufactured by SIGMA-ALDRICH) dissolved in distilled water to give 1.5 mg / mL 30 minutes after LH administration on the 7th day of LH administration was 0.5 mg / mL as LPS.
- Intracerebroventricular administration was performed so that the body weight was kg.
- mice were euthanized, and cerebral cortex and hippocampus were collected.
- 10 ⁇ L of distilled water was administered into the ventricle instead of LPS, and tissues were collected in the same manner as in the above 3 groups.
- TNF- ⁇ the amount of TNF- ⁇ in the sampled tissue was evaluated. That is, the cerebral cortex and hippocampus collected were crushed in RIPA buffer (manufactured by WAKO), and TNF- ⁇ was quantified using an ELISA kit (“Mouse TNF alpha ELISA Ready-SET-Go!” EBioscience). did. The obtained TNF- ⁇ quantitative value was divided by the total protein concentration in the crushed liquid quantified by the BCA method to obtain the TNF- ⁇ content per unit protein mass.
- the outline of the test schedule is shown in Fig. 3, and the results are shown in Fig. 4, respectively.
- the result was shown by the average value +/- standard error of the measured value in each group.
- the TMG- ⁇ amount tended to be lower in the cerebral cortex and hippocampus in the 50 mg group, and the difference between the LH0 mg group and the 50 mg group in the hippocampus was significant. This revealed that orally administered LH suppressed the inflammatory action in the brain.
- LH which was confirmed to exert an inflammation-inhibiting action in the brain in Test Example 4, was evaluated using animals to determine whether it exerts a stress mitigating / suppressing effect.
- a social interaction test using a stress imparting model confirmed the effect of alleviating the stress state and improving motivation and vitality by LH administration.
- mice Since mice originally have the property of showing social exploration behavior when placed in a chamber containing new individuals, they will mainly stay near the place where the new individual (Aggressor) exists (new individual cage) The most distant area (Avoidance Zone) from the new individual (Aggressor) will not stay (Vaishnav Krishanan., Et al Cell, 2007, Vol.131 (2), p391-404). However, since stressed mice have increased anxiety and reduced motivation for new individuals (Aggressor) ⁇ ⁇ , staying in the vicinity of the new individual (Aggressor) is avoided, and the residence time in Avoidance Zone increases.
- test diet AIN93-G (made by Oriental Yeast) feed (hereinafter referred to as “test diet”) prepared by adding LH to a final concentration of 0.1% (W / W) for 7 days before the start date of stress application. ”) Freely, and continued until the day after the end of stress application (end date of evaluation of exploratory behavior).
- test diet prepared by adding LH to a final concentration of 0.1% (W / W) for 7 days before the start date of stress application. ”) Freely, and continued until the day after the end of stress application (end date of evaluation of exploratory behavior).
- ND group and the control diet group the AIN93-G diet to which LH was not added was freely fed from the same day that the LH diet group started taking the test diet (in the ND group, the LH group and the control diet group). The first day of stress application was defined as “Day 1”).
- the LH diet group and the control diet group were stressed by living together with ICR (CD-1) mice (made by Charles River Japan Co., Ltd.) as an Aggressor once a day for 10 minutes from the stress application start date.
- the exploratory behavior evaluation was carried out on the first, second, fourth, eighth, and eleventh days when each group was stressed.
- the search behavior evaluation is a test for evaluating the search behavior of the test animal with respect to a new individual (Aggressor).
- An outline of the test apparatus is shown in FIG. 5C.
- the search behavior evaluation on day -1 evaluates the search behavior of the test animal in the absence of a new individual (Aggressor).
- FIG. 6A shows the change over time of the stay time in the avoidance zone in each group.
- the staying time is shown as an average value ⁇ standard error of measured values of each group.
- the group to which stress was applied (Defeated group; LH group and control food group) tended to have a greater degree of increase over time.
- the LH food group tended to suppress the degree of increase over time.
- the stay time of Avoidance Zone on the second, fourth, eighth and eleventh days of stress application is shown in FIG. 6B.
- the staying time is shown as an average value ⁇ standard error of each group.
- the residence time at Avoidance Zone tended to be shorter in the LH diet group than in the control diet group.
- the difference between the time spent in the Avoidance Zone in the control diet group and that in the LH diet group was significant.
- Test Example 6 It was investigated whether the dipeptide in which the effect of having high anti-inflammatory activity of microglia was found in Test Example 1 and the oligopeptide containing the dipeptide were contained in the sequence of the protein used in food. Specifically, it was investigated whether the sequences of LH, DV, MH, LHL, NLH, TDVE, and NLHL appear in the sequences of 34 types of proteins shown in Table 2 below.
- Table 3 shows the results of appearance of dipeptide sequences, tripeptides, and tetrapeptides for casein protein, whey protein, and BSA, respectively. Since ⁇ -Casein and proteose peptone are degradation products of ⁇ -Casein, oligopeptides that have been confirmed to appear in ⁇ -Casein may also appear in ⁇ -Casein and proteose peptone. Conceivable.
- Table 4 shows the results of appearance of dipeptides, tripeptides and tetrapeptides for glycinin koji, ⁇ -conglycinin, gliadin, glutenin, and lipoviterin, respectively.
- Table 5 shows the results of appearance of dipeptides, tripeptides and tetrapeptides for ovalbumin, ovotransferrin, ovomucoid, ovomucin (mucin 5B), ovomucin (mucin 6), lysozyme, and collagen, respectively.
- the food-derived protein shown above contains at least one core dipeptide. Further, tripeptides and tetrapeptides confirmed to have inflammation suppressing activity in Test Example 3 are included in ⁇ -casein and the like. By using the oligopeptide contained in the protein contained in these foods, a peptide composition having a high anti-inflammatory effect of microglia can be produced.
- ⁇ Test Example 7> Anti-inflammatory effect of microglia by water extract of shochu Addition of rice bran (white rice cake or black rice cake) to steamed raw material (white rice (uruchi rice), barley or sweet potato) according to conventional methods, and cooled I made it. Yeast was added here and fermented. Furthermore, the main raw material (white rice (uruchi rice), barley or satsuma koji) which was cooled after steaming was added and further fermented. The mash obtained by fermentation was distilled to produce shochu raw sake.
- rice bran white rice cake or black rice cake
- steamed raw material white rice (uruchi rice), barley or sweet potato
- Table 8 shows a sample.
- the anti-inflammatory effect when a freeze-dried sample was added was evaluated. Specifically, freeze-dried samples were added to culture plates seeded with purified primary cultured microglia at final concentrations of 0.1, 0.3, 1 and 3 mg / mL, respectively, and cultured for 24 hours. Culturing was performed by adding IFN- ⁇ to final concentrations of 5 ng / mL and 0.5 ng / mL, respectively (0.1 mg group, 0.3 mg group, 1 mg group, 3 mg group). And the quantity of TNF- (alpha) in the culture supernatant 12 hours after culture
- a system in which LPS and IFN- ⁇ are added without adding a lyophilized sample is referred to as a control (+) group, and a system in which neither LPS nor IFN- ⁇ is added is referred to as a control ( ⁇ ) group.
- the result is shown in FIG. 7A.
- the result is the average value of the duplicate data.
- Results are shown in FIG. 7B.
- the tendency for the amount of TNF- ⁇ to be small was recognized as compared with that before filtration (FIG. 7A), and it was found that there was an inflammation suppressing effect equivalent to or higher than that before filtration. From this, it was found that the involved component was contained in a fraction of 2 kDa or less.
- LH concentration in freeze-dried sample LH concentration was quantified by LC / MSMS method. That is, an analysis sample obtained by ultrafiltration (10 kDa) of the supernatant of the lyophilized sample dissolved in water and centrifuged was appropriately diluted and measured by LC / MSMS under the following analysis conditions. Concentration conversion was based on a calibration curve method.
- Mass spectrometer 4000Q TRAP (manufactured by AB Scientific) Pump: Agilent 1200 Binary Pump (manufactured by Agilent Technologies) Autosampler: Agilent 1200 High Performance Autosampler (manufactured by Agilent Technologies) Software version: Analyst 1.6.2 Column: TSK ge1 ODS-100V 3 ⁇ m 2.0 mm ID ⁇ 150 mm (manufactured by Tosoh Corporation) Column temperature: 70 ° C Mobile phase A: 0.1 vol% formic acid Original mobile phase B: 0.1 vol% formic acid / acetonitrile
- the results are shown in Table 11.
- the concentration of LH is calculated as the dry mass of the shochu water extract. Even when any one of glutinous rice, barley, and sweet potato was used as the main raw material, it was confirmed that LH was produced by fermentation, indicating that LH is involved in the anti-inflammatory action of microglia. Moreover, it is confirmed that LH is produced by fermentation, regardless of whether white rice or black rice cake is used as the koji mold, even if any of glutinous rice, barley, and sweet potato is used as the koji raw material. It was shown to be involved in the action.
- shochu preferably shochu water extract
- shochu contains an ingredient that has an effect of relieving stress and an effect of suppressing vitality reduction and motivation / motivation maintenance.
- Shochu is known not only to be eaten by humans but also to animals as feed. Therefore, it was found that the use of shochu can be expected to relieve stress on humans, livestock and pets.
- Preparation Example 1 A skim milk powder or a defatted soybean protein is dissolved in water and subjected to an enzyme treatment to produce a peptide extract containing about 1% of LH, DV or MH. Using this peptide extract, a lemon-flavored carbonated beverage containing LH, DV or MH having the composition shown in Table 13 is prepared. In addition, LH, DV, and MH in the peptide extract and beverage can be quantified by the LC / MSMS method shown in Test Example 7, respectively.
- LH, DV, or MH can be effectively ingested by ingesting this lemon-flavored carbonated beverage.
- Preparation Example 2 Similar to Preparation Example 1, a peptide extract containing about 1% of LH, DV or MH is produced. Using this peptide extract, a coffee preparation liquid containing LH, DV or MH having the composition shown in Table 14 is prepared and sterilized at 121 ° C. for 10 minutes to obtain a canned coffee drink. In addition, LH, DV, and MH in the peptide extract and beverage can be quantified by the LC / MSMS method shown in Test Example 7, respectively.
- LH, DV, or MH can be effectively ingested by ingesting this coffee beverage.
- Preparation Example 3 Similar to Preparation Example 1, a peptide extract containing about 1% of LH, DV or MH is produced. Using this peptide extract, a milk tea preparation solution containing LH, DV or MH having the composition shown in Table 15 is prepared and sterilized at 121 ° C. for 10 minutes to obtain a canned milk tea beverage. In addition, LH, DV, and MH in the peptide extract and beverage can be quantified by the LC / MSMS method shown in Test Example 7, respectively.
- LH, DV or MH can be effectively ingested by ingesting this milk tea beverage.
- Preparation Example 4 Similar to Preparation Example 1, a peptide extract containing about 1% of LH, DV or MH is produced. Using this peptide extract, a fruit juice drink containing LH, DV or MH having the composition shown in Table 16 is prepared. In addition, LH, DV, and MH in the peptide extract and beverage can be quantified by the LC / MSMS method shown in Test Example 7, respectively.
- LH, DV, or MH can be effectively ingested by ingesting this fruit juice drink.
- Preparation Example 5 Similar to Preparation Example 1, a peptide extract containing about 1% of LH, DV or MH is produced. Using this peptide extract, a sports drink beverage containing LH, DV, or MH having the composition shown in Table 17 is prepared. In addition, LH, DV, and MH in the peptide extract and beverage can be quantified by the LC / MSMS method shown in Test Example 7, respectively.
- LH, DV or MH can be effectively ingested by ingesting this sports drink beverage.
- a peptide extract containing about 1% of LH, DV or MH is produced.
- a yogurt flavored beverage containing LH, DV or MH having the composition shown in Table 18 is prepared.
- raw materials other than the starter are homogenized at about 70 ° C. (15 MPa), sterilized by heating (95 ° C. for 15 minutes), added with a starter, and fermented at 30 ° C. for 10 hours.
- the pH reaches 4.60 the mixture is stirred, cooled to around 25 ° C., and emulsified (15 MPa).
- LH, DV, and MH in the peptide extract and beverage can be quantified by the LC / MSMS method shown in Test Example 7, respectively.
- LH, DV or MH can be effectively ingested by ingesting this yogurt flavored beverage.
- a peptide extract containing LH, DV or MH, or a synthetic dipeptide of LH, DV or MH is produced.
- This peptide extract or synthetic dipeptide is used to produce tablets containing LH, DV or MH.
- Cellulose, cyclic oligosaccharide, sucrose ester, paste (pullulan), calcium phosphate, etc. can be blended in the raw material. By taking this tablet, LH, DV or MH can be taken effectively.
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Abstract
Description
また、健康食品および機能性食品、好適にはミクログリアの炎症抑制作用、気分障害抑制作用、またはストレス緩和効果の発揮を意図した成分を含有する食にさらに含有させることもできる。ミクログリアの炎症抑制作用、気分障害抑制作用、またはストレス緩和効果の発揮を意図した成分とは、たとえばテアニン、ポリフェノール類、ローズマリー加水分解産物、ユビデカレノン、エゾウコギ、アクティン、乳酸菌発酵酸乳、βユーデスモールなどである。
ミクログリアの炎症性サイトカイン産生に及ぼすオリゴペプチドの影響を網羅的に調べるために、まず、ジペプチドを合成し以下の試験を行った。
タンパク質を構成する20種類のアミノ酸のうちシステイン(C)を除く19種類のアミノ酸で構成され得るジペプチドの組み合わせ361通りのうち、そのN末端アミノ酸(第一アミノ酸)及びC末端アミノ酸(第二アミノ酸)の組合わせとして、下記表1に示すように、25種のジペプチドを除く336通りの組合せのジペプチドを準備した。なお、立体異性体がある場合はL体が用いられた。
ミクログリアは、マウス脳から磁気細胞分離法によって単離した。具体的には、マウスより摘出した脳をパパイン処理することにより脳組織分散液を得、酵素反応を停止させた。次に、超常磁性マイクロビーズによって磁性標識された汎ミクログリアマーカーであるCD11b抗体(Miltenyi Biotec社製)と反応させ、それを磁気分離することにより、
ミクログリアを単離した。
単離したミクログリア細胞を、上記336種のジペプチドを各々50μM濃度で添加した培地で12時間培養した。その後、5ng/ml lipopolyshccaride (LPS、SIGMA-ALDRICH社製)及び0.5ng/ml IFN-γ(R&D system社製)を添加して、さらに12時間培養し、培養上清中に含まれるTNF-αをELISAキット(eBioscience社製)を用いて定量した。ペプチドを添加していないウェルをコントロールとし、コントロールを1としたときのTNF-αの産生割合を算出した。結果を表1に示す。なお、結果は3連のデータの平均値である。また、以下、アミノ酸は一文字略号で示す。
試験例1で優れた抗炎症効果が認められたジペプチドのうち、LH、DVについてさらに解析を行った。まず、これらジペプチドの抗炎症作用が濃度依存的であるか解析を行った。
次に、これらジペプチドがコア配列として機能するか解析するために、DV、LHをコア配列として有するトリペプチド、テトラペプチドのミクログリアに対する炎症抑制効果の解析を行った。結果を図2に示す。2種のペプチドDV、LHをコア配列として有するトリペプチド、DVE、TDV、LHL、NLH、テトラペプチドTDVE、NLHLについて抗炎症作用を解析した。
LH投与によって脳内の炎症が緩和されることを確認した。
6週齢のICR(CD-1)雄マウス(日本チャールズリバー社製)を4群に分けた。LH0mg群4匹、LH10mg群3匹、LH50mg群5匹にはそれぞれ、0、10、50mg/kg体重となるように調製したLH(国産化学社製)を1日1回、7日間連続
で胃内へ強制経口投与した。
(1)社会的相互作用試験
ストレスを受け、それに伴い慢性疲労や意欲低下、抑うつの状態が見られることがあるが、メカニズムとして脳内の炎症が関与していることが近年の研究により明らかとなっている(古屋敷智之“心理ストレスにおける炎症関連分子の役割とミクログリア活性化への関与”実験医学Vol.30-No.13 2012, 65-71)。
試験概要を図5A~図5Cに示す。
探索行動評価は新規個体(Aggressor)に対する試験動物の探索行動を評価する試験である。試験装置の概略は図5Cに示す。縦40cm、横30 cm、高さ30cmのチャンバーの壁際にケージに入った新規個体(Aggressor)であるICR(CD-1)マウスを置いたうえで該チャンバー内に試験動物を置き、5分間(300秒間)自由に探索させ、Avoidance Zoneにおける滞在時間を計測した。なお、-1日目の探索行動評価は、新規個体(Aggressor)不在状況下での、試験動物の探索行動を評価している。
各群におけるAvoidance Zoneでの滞在時間の経時変化を図6Aに示した。滞在時間は各群の測定値の平均値±標準誤差として示している。各群ともAvoidance Zone滞在時間が経時的に増加する傾向が認められた。ND群と比較して、ストレスを付与した群(Defeated群;LH群およびコントロール食群)において経時的増加の程度が大きい傾向にあった。また、コントロール食群と比較して、LH食群においては、経時的増加の程度が抑制される傾向にあった。
試験例1でミクログリアの高い炎症抑制活性を有する効果が見出されたジペプチド及び該ジペプチドを含むオリゴペプチドが、食品に用いられるタンパク質の配列中に含まれているかを調べた。具体的には、下記表2に示す34種類のタンパク質の配列中に、LH、DV、MH、LHL、NLH、TDVE、NLHLの配列が現れるかを調べた。
(1)焼酎粕の水抽出物による、ミクログリアの炎症抑制効果
常法にしたがって麹原料(白米(うるち米)、大麦又はさつま芋)を蒸煮、冷却したものに種麹(白麹又は黒麹)を添加し製麹した。ここに酵母を加えて発酵させた。さらに、蒸煮のうえ冷却した主原料(白米(うるち米)、大麦又はさつま芋)を加えさらに発酵させた。発酵で得られたもろみを蒸留して焼酎原酒を製造した。
(1)で得られた凍結乾燥サンプルを、終濃度としていずれも0.3、1、3mg/mLとなるように加えて調製した培養液を2kDaの限外濾過膜で濾過し、(1)と同様の方法でミクログリアの炎症抑制効果を評価した。
LC/MSMS法によりLH濃度を定量した。すなわち、上記凍結乾燥サンプルを水に溶解し遠心分離したものの上清を限外濾過(10kDa)して得られた分析サンプルを適宜希釈し、以下の分析条件でLC/MSMSにて測定した。濃度換算は検量線法によった。
質量分析計: 4000Q TRAP(エービー・サイエックス社製)
ポンプ: Agilent 1200 Binary Pump(アジレント・テクノロジー社製)
オートサンプラ:Agilent 1200 High Performance Autosampler(アジレント・テクノロジー社製)
ソフトウェアバージョン:Analyst 1.6.2
カラム: TSK ge1 ODS-100V 3 μm 2.0 mm I.D.×150 mm(東ソー社製)
カラム温度: 70℃
穆動相A: 0.1 vol%ギ酸
稿動相B: 0.1 vol%ギ酸/アセトニトリル
スキャンタイプ:MRM(MRM)
極性: 陽性
スキャンモード:N/A
イオン源: ターボスプレー
分解能 Q1: ユニット
分解能 Q3: ユニット
強度閾値: 0.00 cps
セトリング時間:0.0000 msec
MRポーズ: 5.0000 msec
MCA: なし
ステップサイズ:0.00 Da
上記主原料中および一般に焼酎原料として汎用されるジャガイモのタンパク質にLHなるアミノ酸配列が含まれるかウェブサイト情報から予測した。結果は表12に示すとおりである。主原料として使用する大麦、米、さつま芋、ジャガイモのいずれにおいてもLH配列を含むタンパク質が存在することが確認され、これらを発酵させることでLHが生成する可能性が、文献的にも示された。
焼酎粕(好ましくは焼酎粕水抽出物)が、ミクログリアの炎症を抑制する効果を有することが明らかとなった。試験例4及び5の結果も併せて考えると、焼酎粕にはストレスを緩和する効果を有し、活力低下の抑制効果や意欲・モチベーション維持効果を有する成分が含まれることが推測された。焼酎粕はヒトが食することはもちろん、飼料などとして動物も食することが知られている。そのため、焼酎粕の利用によりヒトや家畜、ペットなどのストレスの緩和などが期待できることがわかった。
脱脂粉乳又は脱脂大豆たんぱく質を水に溶解し酵素処理を施し、LH、DV又はMHを約1%含有するペプチドエキスを製造する。このペプチドエキスを使用して、表13に示す組成のLH、DV又はMHを含有するレモン風味炭酸飲料を調製する。なお、ペプチドエキス及び飲料中のLH、DV及びMHはそれぞれ試験例7に示したLC/MSMS法で定量することができる。
調製例1と同様に、LH、DV又はMHを約1%含有するペプチドエキスを製造する。このペプチドエキスを使用して、表14に示す組成のLH、DV又はMHを含有するコーヒー調製液を調製し、121℃で10分間殺菌し缶入りコーヒー飲料とする。なお、ペプチドエキス及び飲料中のLH、DV及びMHはそれぞれ試験例7に示したLC/MSMS法で定量することができる。
調製例1と同様に、LH、DV又はMHを約1%含有するペプチドエキスを製造する。このペプチドエキスを使用して、表15に示す組成のLH、DV又はMHを含有するミルクティ調製液を調製し、121℃で10分間殺菌し缶入りミルクティ飲料とする。なお、ペプチドエキス及び飲料中のLH、DV及びMHはそれぞれ試験例7に示したLC/MSMS法で定量することができる。
調製例1と同様に、LH、DV又はMHを約1%含有するペプチドエキスを製造する。このペプチドエキスを使用して、表16に示す組成のLH、DV又はMHを含有する果汁飲料を調製する。なお、ペプチドエキス及び飲料中のLH、DV及びMHはそれぞれ試験例7に示したLC/MSMS法で定量することができる。
調製例1と同様に、LH、DV又はMHを約1%含有するペプチドエキスを製造する。このペプチドエキスを使用して、表17に示す組成のLH、DV又はMHを含有するスポーツドリンク飲料を調製する。なお、ペプチドエキス及び飲料中のLH、DV及びMHはそれぞれ試験例7に示したLC/MSMS法で定量することができる。
調製例1と同様に、LH、DV又はMHを約1%含有するペプチドエキスを製造する。このペプチドエキスを使用して、表18に示す組成のLH、DV又はMHを含有するヨーグルト風味飲料を調製する。その調製は、スターター以外の原料を、約70℃でホモジナイズし(15MPa)、加熱殺菌(95℃15分)後、スターターを添加し、30℃で10時間発酵させる。pHが4.60に達した時点で攪拌し、25℃付近まで冷却し、乳化(15MPa)処理をする。なお、ペプチドエキス及び飲料中のLH、DV及びMHはそれぞれ試験例7に示したLC/MSMS法で定量することができる。
LH、DV又はMHを含有するペプチドエキス、又はLH、DV又はMHの合成ジペプチドを製造する。このペプチドエキス又は合成ジペプチドを使用して、LH、DV又はMHを含有するタブレットを製造する。原料にはセルロース、環状オリゴ糖、ショ糖エステル、糊料 (プルラン)、リン酸カルシウム等を配合することができる。このタブレットを摂取することにより、LH、DV又はMHを有効に摂取することができる。
Claims (12)
- LH、DVもしくはMHで表されるアミノ酸配列を有するジペプチド、又は該アミノ酸配列をコア配列として含むオリゴペプチド、又はその薬学上許容される塩もしくは溶媒和物を含む、ミクログリアの炎症を抑制するための組成物。
- LH、DVもしくはMHで表されるアミノ酸配列を有するジペプチド、又は該アミノ酸配列をコア配列として含むオリゴペプチド、又はその薬学上許容される塩もしくは溶媒和物を含む、慢性疲労症候群、認知症及び/又は気分障害の症状を緩和、治療、又は予防するための組成物。
- LH、DVもしくはMHで表されるアミノ酸配列を有するジペプチド、又は該アミノ酸配列をコア配列として含むオリゴペプチド、又はその薬学上許容される塩もしくは溶媒和物を含む、ストレスにより引き起こされる状態を緩和又は予防するための組成物。
- LHで表されるアミノ酸配列を有するジペプチド又はその薬学上許容される塩もしくは溶媒和物を含む、ストレスにより引き起こされる状態を緩和又は予防するための組成物。
- 前記ストレスにより引き起こされる状態が、意欲・モチベーションの欠如、及び/又は活力の低下である、請求項3又は請求項4記載の組成物。
- 飲食品組成物である請求項1乃至請求項5いずれか1項記載の組成物。
- 医薬組成物である請求項1乃至請求項5いずれか1項記載の組成物。
- タンパク質を加水分解して、LH、DVもしくはMHで表されるアミノ酸配列を有するジペプチド、又は該アミノ酸配列をコア配列として含むオリゴペプチド、又はその薬学上許容される塩もしくは溶媒和物を得る工程を含むことを特徴とする、請求項1乃至請求項7いずれか1項記載の組成物の製造方法。
- 前記、タンパク質を加水分解して、LH、DVもしくはMHで表されるアミノ酸配列を有するジペプチド、又は該アミノ酸配列をコア配列として含むオリゴペプチド、又はその薬学上許容される塩もしくは溶媒和物を得る工程により得られた組成物を、精製・濃縮する工程をさらに含むことを特徴とする、請求項8記載の方法。
- 前記、タンパク質を加水分解して、LH、DVもしくはMHで表されるアミノ酸配列を有するジペプチド、又は該アミノ酸配列をコア配列として含むオリゴペプチド、又はその薬学上許容される塩もしくは溶媒和物を得る工程により得られた組成物が、酒粕であることを特徴とする、請求項8又は請求項9記載の方法。
- LH、DVもしくはMHで表されるアミノ酸配列を有するジペプチド、又は該アミノ酸配列をコア配列として含むオリゴペプチド、又はその薬学上許容される塩もしくは溶媒和物を含有する酒粕を含有させる工程を含むことを特徴とする、ストレスにより引き起こされる状態を緩和又は予防するための組成物の製造方法。
- 前記、LH、DVもしくはMHで表されるアミノ酸配列を有するジペプチド、又は該アミノ酸配列をコア配列として含むオリゴペプチド、又はその薬学上許容される塩もしくは溶媒和物を含有する酒粕を、精製・濃縮する工程をさらに含むことを特徴とする、請求項11記載の方法。
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CN112041328A (zh) * | 2018-04-26 | 2020-12-04 | 志瑞亚新药工业株式会社 | 二肽和含有该二肽的药物组合物 |
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CN112041328B (zh) * | 2018-04-26 | 2024-06-21 | 志瑞亚新药工业株式会社 | 二肽和含有该二肽的药物组合物 |
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