Composition for improving blood hemoglobin level

Abstract

To provide new means for improving hemoglobin levels in blood.SOLUTION: The present invention provides a composition for improving hemoglobin levels in blood, a composition for improving erythrocyte count in blood, a composition for promoting erythropoietin production, or a composition for preventing or improving anemia, each comprising, as an active ingredient, at least one selected from the group consisting of milk-derived peptides, derivatives thereof, and salts thereof.SELECTED DRAWING: Figure 3

Description

Technical Field

[0001] The present invention relates to a composition for improving the amount of hemoglobin in blood.

Background Art

[0002] Anemia, as defined by the WHO, refers to a condition where the hemoglobin level in the blood is 13.0 g / dL or less in men and 12.0 g / dL or less in women, and may be accompanied by shortness of breath, dizziness, and fatigue. In anemia or its pre-stage condition, since oxygen transport by hemoglobin is insufficient, in addition to a decrease in exercise performance and intellectual activity, discomfort associated with systemic oxygen deficiency may occur.

[0003] As causes of a decrease in hemoglobin in blood without accompanying diseases or bleeding, in addition to iron deficiency, hemolytic anemia (so-called "sports anemia") in which red blood cells are destroyed by exercise that gives a strong impact to the soles of the feet is known. As a pre-stage of sports anemia, an "iron deficiency state without anemia" (IDNA) is known in which the hemoglobin level is normal but the blood concentration of ferritin, a protein that stores iron supplied to the blood in the body, decreases. IDNA is often defined as a case where the serum ferritin concentration is less than 30 μg / L in men and less than 20 μg / L in women (see Non-Patent Document 1).

[0004] As one of the means for improving such anemia and its pre-stage condition, in addition to supplying iron, administration of erythropoietin (EPO) is performed. EPO is a peptide hormone and promotes the differentiation of hematopoietic stem cells in the bone marrow into red blood cells. A decrease in the production of EPO is known to cause anemia.

[0005] On the other hand, Patent Document 1 reports that a peptide having an amino acid sequence consisting of KHP (SEQ ID NO: 1), KIHP (SEQ ID NO: 6), or HKEMPFPKY (SEQ ID NO: 20), or a derivative thereof, or a salt thereof has excellent safety and an interleukin-12 production-inducing action.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Non-Patent Documents

[0007]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0008] As described above, in order to improve anemia or its pre-stage state, it is important to maintain the amount of hemoglobin in the blood at a normal level. However, the application of EPO is limited to diseases and requires administration by injection, and it is a target of doping in sports competitions, so it is not suitable for daily anemia or risk countermeasures in its pre-stage.

[0009] Therefore, an object of the present invention is to provide a new means for improving the amount of hemoglobin in the blood.

Means for Solving the Problems

[0010] As a result of intensive studies, the present inventors have found that a composition containing a specific milk-derived peptide promotes the production of erythropoietin, improves the amount of hemoglobin, and improves anemia or its pre-stage state, and thus completed the present invention.

[0011] That is, the present invention includes, but is not limited to, the following aspects. [1] A composition for improving the amount of hemoglobin in the blood, containing at least one selected from the group consisting of a milk-derived peptide, its derivative, and their salts as an active ingredient. [2] A composition for improving the number of red blood cells in the blood, containing at least one selected from the group consisting of milk-derived peptides, their derivatives, and their salts as an active ingredient. [3] A composition for promoting the production of erythropoietin, containing at least one selected from the group consisting of milk-derived peptides, their derivatives, and their salts as an active ingredient. [4] A composition for preventing or improving anemia, containing at least one selected from the group consisting of milk-derived peptides, their derivatives, and their salts as an active ingredient. [5] The composition according to any one of [1] to [4] for improving any one of the following (a) to (d): (a) Poor color or pale eyelid conjunctiva, (b) Headache, dizziness, or lightheadedness, (c) Fatigue, lassitude, or weakness, (d) Shortness of breath or increased heart rate. [6] The composition according to any one of [1] to [5], which is administered or ingested to any of the following individuals: (a) Individuals with iron deficiency without anemia (IDNA), (b) Individuals who perform high-load exercise daily, (c) Women. [7] The composition according to any one of [1] to [6], comprising a peptide consisting of at least one amino acid sequence selected from the group consisting of the milk-derived peptides KHP (SEQ ID NO: 1), VRY (SEQ ID NO: 2), DIK (SEQ ID NO: 3), KEK (SEQ ID NO: 4), IKHQ (SEQ ID NO: 5), KIHP (SEQ ID NO: 6), SRYP (SEQ ID NO: 7), RYPS (SEQ ID NO: 8), KYIP (SEQ ID NO: 9), IHPF (SEQ ID NO: 10), MKPW (SEQ ID NO: 11), HQPHQ (SEQ ID NO: 12), VEQKH (SEQ ID NO: 13), KDERF (SEQ ID NO: 14), VDDKHY (SEQ ID NO: 15), DDKHYQ (SEQ ID NO: 16), KYKVPQ (SEQ ID NO: 17), VDDKHYQ (SEQ ID NO: 18), VDDKHYQK (SEQ ID NO: 19), and HKEMPFPKY (SEQ ID NO: 20).

Advantages of the Invention

[0012] In addition, by administering the composition of the present invention to a subject, it is possible to promote the production of erythropoietin, improve the hemoglobin level, and improve anemia or a state prior to anemia.

Brief Description of the Drawings

[0013]

Fig. 1A

Fig. 1B

Fig. 2

Fig. 3

Fig. 4

Mode for Carrying Out the Invention

[0014] The composition of the present application contains at least one selected from the group consisting of milk-derived peptides, their derivatives, and their salts as an active ingredient.

[0015] (Milk-derived peptide) The "milk-derived peptide" which is the active ingredient is a mixture of peptides produced by hydrolyzing proteins derived from mammalian milk, but its origin is not particularly limited. Examples of mammalian milk include cow's milk, goat milk, sheep milk, human milk, etc., and cow's milk is preferred. The milk-derived peptide is not particularly limited, but can be prepared by, for example, any of the following methods (i) to (iii).

[0016] <Mode (i) Preparation by hydrolysis of milk-derived protein> (i) In one embodiment, the milk-derived peptide is prepared by hydrolyzing milk-derived protein. This preparation method is particularly preferred because it is low-cost and easy for large-scale production.

[0017] In one embodiment, the milk-derived protein contains at least one selected from the group consisting of skim milk powder, whey protein, milk protein concentrate (MPC), and casein.

[0018] Operations such as hydrolysis of milk-derived proteins can be carried out, for example, according to the method described in the publication with international publication number WO89 / 06970. Hydrolysis is carried out, for example, using one or more hydrolytic enzymes such as acidic protease, neutral protease or alkaline protease. In one embodiment, the milk-derived protein is hydrolyzed using neutral protease, neutral peptidase or both.

[0019] When hydrolyzing milk-derived proteins with neutral protease, neutral peptidase, or both, the reaction conditions used are not particularly limited and can be appropriately selected by those skilled in the art according to common general knowledge. As the pH of the reaction solution, pH 5 to 8.5 is preferred, and pH 6.5 to 7.5 is more preferred. The temperature can be appropriately changed according to the optimum temperature of the enzyme used, but it may be adjusted so that the peptide is contained in the molecular weight distribution described below.

[0020] There are also no particular restrictions on the means for stopping the reaction, and known means can be used. Examples of such means include heat treatment (for example, 70°C or higher, 80°C or higher, or 90°C or higher).

[0021] From the hydrolyzate of milk-derived proteins, a water-insoluble fraction (for example, insoluble proteins including inactivated enzymes, etc.) may be removed by filtration or the like by a known method as appropriate. For example, when the enzyme is inactivated by heating, the water-insoluble fraction is preferably removed by filtration or adsorption. For filtration or adsorption, for example, activated carbon, diatomaceous earth, silicon dioxide, kaolin, acid clay, perlite, bentonite, zeolite, talc, sand, powdered cellulose, etc. can be used.

[0022] <Aspect (ii) Preparation by chemical synthesis> (ii) In another embodiment, the milk-derived peptide is chemically synthesized from among the peptides obtained in aspect (i) by a known method (for example, solid-phase peptide synthesis using a protecting group such as a tert-butyloxycarbonyl group or a 9-fluorenylmethyloxycarbonyl group, liquid-phase method, etc.) for a peptide having the action according to the present invention.

[0023] <Aspect (iii) Preparation by a biosynthetic method> (iii) In yet another embodiment, the milk-derived peptide is produced by a biosynthetic method involving the translation of mRNA encoding a peptide having the action according to the present invention among the peptides obtained by the method of aspect (i). The translation of mRNA may be carried out, for example, in various cells (such as bacteria like Escherichia coli, yeast, animal cells, plant cells, etc.) or by an in vitro translation system.

[0024] For the preparation of the milk-derived peptide of the present invention, one kind alone selected from the group consisting of the preparations prepared in each of the above aspects (i) to (iii) and milk-derived peptide-containing compositions obtained by different methods may be used, or alternatively, two or more kinds may be used in combination. For the compositions containing those milk-derived peptides, it is preferable to further fractionate, purify, concentrate, etc. by a known method as appropriate.

[0025] From the viewpoint of increasing the concentration of the active ingredient, it is preferable to fractionate mammalian milk or its fractions (such as skim milk powder, whey, etc.) according to the molecular weight for the preparation containing the milk-derived peptide (particularly the preparation of aspect (i)). For the molecular weight fractionation, for example, ultrafiltration or gel filtration chromatography can be used.

[0026] The weight average molecular weight of the milk-derived peptide obtained by the molecular weight fractionation is preferably 8000 or less, more preferably 2000 or less, still more preferably 200 to 2000, for example, 200 to 1500, 200 to 1000, 1000 to 2000, 500 to 1000 or 200 to 500, from the viewpoint of significantly exhibiting the effect of the present invention and having high water solubility.

[0027] In the present specification, the weight average molecular weight of the peptide is measured by the gel permeation chromatography (GPC) method.

[0028] In the method for producing milk-derived peptides, depending on the production method, form, use, etc., a concentration step, a drying step, a sterilization step, a packaging step, etc. can be further included. In the drying step, a spray drying method, a drum drying method, a freeze drying method, a vacuum drying method, etc. can be used. In one embodiment, the method for producing milk-derived peptides includes a drying step by the spray drying method.

[0029] The content of peptides with a molecular weight of 1000 - 3000 per 1 part by mass of peptides with a molecular weight of 200 - 1000 in the milk-derived peptides used in the present invention is preferably 0.005 - 15 parts by mass, more preferably 0.01 - 10 parts by mass, still more preferably 0.02 - 5 parts by mass, even more preferably 0.05 - 3 parts by mass, particularly preferably 0.1 - 1.5 parts by mass, and particularly more preferably 0.2 - 0.6 parts by mass.

[0030] The content of peptides with a molecular weight of 1000 - 2000 per 1 part by mass of peptides with a molecular weight of 500 - 1000 in the milk-derived peptides used in the present invention is preferably 0.01 - 10 parts by mass, more preferably 0.02 - 5 parts by mass, still more preferably 0.05 - 3 parts by mass, even more preferably 0.1 - 2 parts by mass, particularly preferably 0.2 - 1 part by mass, and particularly more preferably 0.4 - 0.8 parts by mass.

[0031] The content of peptides with a molecular weight of 200 - 500 per 1 part by mass of the content of peptides with a molecular weight of 500 - 1000 in the milk-derived peptides used in the present invention can be preferably 0.02 - 20 parts by mass, more preferably 0.05 - 15 parts by mass, still more preferably 0.1 - 10 parts by mass, even more preferably 0.2 - 5 parts by mass, particularly preferably 0.4 - 3 parts by mass, and particularly more preferably 0.6 - 1.5 parts by mass.

[0032] The content of peptides with a molecular weight of 2,000 to 3,000 per part by mass of the content of peptides with a molecular weight of 500 to 1,000 in the milk-derived peptides used in the present invention is preferably 0.002 to 10 parts by mass, more preferably 0.005 to 5 parts by mass, still more preferably 0.01 to 3 parts by mass, even more preferably 0.02 to 1.5 parts by mass, particularly preferably 0.05 to 0.8 parts by mass, and particularly more preferably 0.1 to 0.4 parts by mass.

[0033] In one embodiment, the composition of the present invention contains milk-derived peptides, derivatives thereof, and salts thereof, and the milk-derived peptides include any one peptide consisting of an amino acid sequence selected from the group consisting of KHP (SEQ ID NO: 1), VRY (SEQ ID NO: 2), DIK (SEQ ID NO: 3), KEK (SEQ ID NO: 4), IKHQ (SEQ ID NO: 5), KIHP (SEQ ID NO: 6), SRYP (SEQ ID NO: 7), RYPS (SEQ ID NO: 8), KYIP (SEQ ID NO: 9), IHPF (SEQ ID NO: 10), MKPW (SEQ ID NO: 11), HQPHQ (SEQ ID NO: 12), VEQKH (SEQ ID NO: 13), KDERF (SEQ ID NO: 14), VDDKHY (SEQ ID NO: 15), DDKHYQ (SEQ ID NO: 16), KYKVPQ (SEQ ID NO: 17), VDDKHYQ (SEQ ID NO: 18), VDDKHYQK (SEQ ID NO: 19), and HKEMPFPKY (SEQ ID NO: 20), or a combination of two or more thereof. In this specification, these peptides are referred to as "20 specific peptides".

[0034] The total content of the above 20 specific peptides is preferably 0.001% by mass or more, more preferably 0.01% by mass or more, still more preferably 0.1% by mass or more, and even more preferably 0.2% by mass or more with respect to the total amount of milk-derived peptides, and can be, for example, 99% by mass or less, 90% by mass or less, 70% by mass or less, 50% by mass or less, 20% by mass or less, 10% by mass or less, 5% by mass or less, 2% by mass or less, 1% by mass or less, or 0.5% by mass or less.

[0035] The preferred range of the content of other polypeptides relative to 1 part by mass of KHP (SEQ ID NO: 1) in the milk-derived peptide or in the composition of the present invention is such that each polypeptide can independently take any of the numerical ranges described in Table 2. In one embodiment, the content of the polypeptides of SEQ ID NOs: 2 to 20 relative to 1 part by mass of KHP (SEQ ID NO: 1) in the milk-derived peptide is the combination described in the "preferred range", "more preferred range", "even more preferred range" or "even more preferably range" in Table 1.

[0036]

Table 1

[0037] In the present specification, the salt of the milk-derived peptide is not particularly limited as long as it is a pharmacologically or physiologically acceptable salt, and specifically includes organic acid salts, inorganic acid salts, salts with organic bases, or salts with inorganic bases. Examples of organic acid salts include monocarboxylic acid salts such as acetate, trifluoroacetate, butyrate, palmitate, and stearate; polyvalent carboxylate salts such as fumarate, maleate, succinate, and malonate; oxycarboxylate salts such as lactate, tartrate, and citrate; and organic sulfonate salts such as methanesulfonate, toluenesulfonate, and tosylate. Examples of inorganic acid salts include hydrochloride, sulfate, nitrate, hydrobromide, and phosphate. Examples of salts with organic bases include salts with organic amines such as methylamine, triethylamine, triethanolamine, diethanolamine, morpholine, piperazine, pyrrolidine, tripyridine, picoline, and ethylenediamine. Examples of salts with inorganic bases include various salts such as ammonium salts; salts with metals such as alkali metals such as sodium or potassium, alkaline earth metals such as calcium or magnesium, and aluminum.

[0038] In this specification, a "derivative" of a milk-derived peptide refers to a chemically modified product obtained from a milk-derived peptide through a chemical reaction or a biochemical process (including intracellular processes such as translational modification). Examples of derivatives of milk-derived peptides include milk-derived peptides that have been subjected to, for example, sugar chain modification, lipid modification, PEGylation, N-terminal or C-terminal modification (such as modification that confers peptidase resistance like N-terminal acetylation, C-terminal amidation, etc.).

[0039] (Other components) Depending on the use, form, etc. of the composition of the present invention, in addition to the above milk-derived peptides, for example, bases, carriers, additives, iron (such as heme iron, ferritin, lactoferrin, ferric chloride, sodium ferrous citrate, iron citrate, ammonium iron citrate, ferrous gluconate, iron lactate, ferrous sulfate, ferric pyrophosphate, etc.), citric acid, vitamin C, vitamin B 12 and other components such as folic acid may be further included.

[0040] (Use) As shown in the following examples, the composition of the present invention has an action of promoting an increase in the amount of hemoglobin in the blood, an action of promoting erythropoietin gene expression, an action of promoting IGF-1 gene expression, and an action of promoting HIF-1A gene expression. Therefore, the composition of the present invention is suitable for uses such as improving the amount of hemoglobin in the blood, promoting the expression of erythropoietin (EPO), IGF-1, or HIF-1A genes or promoting the production of their proteins, or uses for improving anemia. Also, as described above, considering that erythropoietin contributes to hematopoiesis promotion by erythrocyte differentiation, the composition of the present invention is also suitable for uses for improving the number of erythrocytes in the blood.

[0041] In this specification, "improvement of the amount of hemoglobin in the blood" more specifically includes maintaining the amount of hemoglobin in the blood within the normal range, promoting an increase in the amount of hemoglobin in the blood (for example, promoting an increase from a blood hemoglobin amount lower than normal to the normal range), suppressing, delaying, or preventing a decrease in the blood hemoglobin value, or reducing the risk of a decrease in the blood hemoglobin value.

[0042] As used herein, "improvement of the red blood cell count in blood" more specifically includes maintaining the red blood cell count in blood within the normal range, increasing the red blood cell count in blood (for example, increasing a red blood cell count lower than normal to the normal range), suppressing, delaying or preventing a decrease in the red blood cell count in blood, or reducing the risk of a decrease in the red blood cell count in blood.

[0043] As used herein, "improvement of anemia" refers to the cure, improvement or remission of anemia or its pre-stage (pre-disease stage); prevention or delay of the exacerbation of anemia or its pre-stage; or reversal, prevention or delay of the progression of anemia or its pre-stage. In one embodiment, the pre-stage (pre-disease stage) of anemia may be iron deficiency without anemia (IDNA). As used herein, IDNA is defined according to the IDNA criteria in athletes, for example, when the serum ferritin concentration is less than 30 μg / L or less than 40 μg / L in males, and less than 20 μg / L or less than 30 μg / L in females.

[0044] Furthermore, since the composition of the present invention improves anemia, it is also suitable for use in improving physical changes or symptoms associated with anemia. Physical changes or symptoms associated with anemia include poor complexion (the complexion becomes pale), pale palpebral conjunctiva, headache, dizziness, fainting, swelling of the feet, fatigue, listlessness, weakness, shortness of breath, increased heart rate, palpitation, shoulder stiffness, skin rash, nail deformation, drowsiness, difficulty in waking up in the morning, and the like. As used herein, "improvement" of physical changes or symptoms associated with anemia refers to the cure, improvement or remission of the physical condition or symptoms; prevention or delay of the exacerbation of the physical condition or symptoms; or reversal, prevention or delay of the progression of the symptoms.

[0045] The composition of the present invention may be used for therapeutic purposes or non-therapeutic purposes. As used herein, "non-therapeutic purposes" refers to uses that do not include medical acts, for example, uses that do not include surgical, therapeutic or diagnostic acts on a subject by a doctor or a person under the instruction of a doctor (for example, uses related to health maintenance or promotion, or beauty).

[0046] (Target Application) The composition of the present invention can be used for vertebrates such as mammals (human or non-human), birds (parrots, crows, pigeons, finches, owls, nightjars, etc.), reptiles (snakes, turtles, lizards, etc.), amphibians (frogs, salamanders, etc.), and fish. In one embodiment, the composition of the present invention is used for human or non-human mammals, preferably humans.

[0047] Examples of non-human mammals include companion animals such as dogs, cats, hamsters, rabbits, ferrets, guinea pigs, etc.; livestock animals such as cows, pigs, horses, goats, sheep, etc.; mice, rats, wild boars, shrews, elephants, armadillos, raccoons, moles, monkeys, chimpanzees, rabbits, degus, squirrels, martens, rats, voles, chinchillas, ferrets, camels, wild boars, deer, elk, bison, whales, dolphins, lions, tigers, wolves, weasels, bears, seals, etc., and more preferably dogs or cats.

[0048] Examples of fish include farmed fish such as yellowtail, red sea bream, eel, tuna, pufferfish, eel, trout, flounder, horse mackerel, mackerel, greater amberjack, ayu, etc.; ornamental fish such as medaka, neon tetra, guppy, etc.; zebrafish, etc.

[0049] The applicable age of the composition of the present invention is not particularly limited. In one embodiment, the composition of the present invention is applied to individuals in the growth period. In individuals in the growth period, the number of red blood cells required increases as the body grows, so the risk of anemia or a state prior to it is relatively high, making it a preferred target.

[0050] Individuals in the growth period of humans can be, for example, 0 years old or older, 3 years old or older, or 6 years old or older, and can be 18 years old or younger, 16 years old or younger, 14 years old or younger, or 12 years old or younger. Examples of individuals in the growth period of humans include, but are not limited to, 0 - 18 years old, 3 - 16 years old, 6 - 14 years old, or 6 - 12 years old.

[0051] In another embodiment, the composition of the present invention is applied to individuals in the mature stage. Human individuals in the mature stage can be, for example, adults, such as 20 years old or older, 25 years old or older, 30 years old or older, 40 years old or older, 50 years old or older, 60 years old or older. Human individuals in the mature stage can be, for example, 20 to 50 years old, 25 to 45 years old, or 25 to 40 years old, but are not limited thereto.

[0052] In one embodiment, the composition of the present invention can be suitably applied to individuals who meet the IDNA criteria corresponding to "iron deficiency without anemia" (IDNA), particularly in athletes. Such individuals can be considered preferred subjects because even if their hemoglobin level is normal, they have an increased potential risk of insufficient hemoglobin levels.

[0053] In one embodiment, the composition of the present invention is applied to individuals who perform high-load exercise. As used herein, high-load exercise (also referred to as high-intensity exercise) is exercise that subjects the soles of the feet to strong impacts such as kicks, jumps, etc., or high weights, and examples include track and field events (e.g., middle-distance running, long-distance running), basketball, volleyball, soccer, kendo, gymnastics competitions, etc. The composition of the present invention is applied to, for example, individuals who perform high-load exercise for 30 minutes or more or 1 hour or more per day. The composition of the present invention is applied to, for example, individuals who perform high-load exercise daily, once every two days, once every three days, or once a week.

[0054] The gender of the subject to which the composition of the present invention is applied is not particularly limited. In humans, women who experience menstruation are potentially at high risk of anemia or a state prior thereto, and are preferred subjects. Examples of women who experience menstruation include, but are not limited to, 10 to 60 years old, 11 to 55 years old, or 12 to 50 years old, etc.

[0055] (Form of the composition) The composition of the present invention can be added to, for example, food and drink products (health foods, supplements, foods with functional claims, foods for patients, foods for specified health uses, etc.), pharmaceuticals, quasi-drugs (drinkable preparations, etc.), feeds, baits, or pet foods and used for their production. Further, the composition of the present invention can also be used, for example, as food and drink products, pharmaceuticals, quasi-drugs, feeds, baits, or pet foods as it is.

[0056] In one embodiment, the composition of the present invention has a product label or package insert of a pharmaceutical or the like that explicitly or implicitly indicates any of the above uses or the effects related thereto. Examples of such compositions include pharmaceuticals, supplements, foods with functional claims, foods for patients, foods for specified health uses, or foods with nutritional functions.

[0057] Examples of labels that explicitly or implicitly indicate the use for preventing or improving anemia include, but are not limited to, combinations such as labels that imply symptoms associated with anemia such as "dizzy" or "faint" and "care" or "support", or labels that imply the use for subjects who have or are at risk of changes in physical condition or symptoms associated with anemia such as "for those who have been told they look pale", "for those who are concerned about dizziness", or "if you feel faint".

[0058] When the composition of the present invention is a food and drink product, its form can be, for example, liquid beverages such as soups, juices, fruit juice drinks, milk, milk drinks, whey drinks, lactic acid bacteria drinks, tea drinks, alcoholic beverages, coffee drinks, carbonated drinks, soft drinks, water drinks, cocoa drinks, jelly-like drinks, sports drinks, diet drinks, etc., semi-solid foods such as puddings, yogurts, etc., noodles such as pasta, ramen, udon, soba, etc., confectioneries, or spreads; or oral preparations (capsules, tablets, chewable tablets, powders, granules, lozenges, liquids, syrups, etc.).

[0059] Pharmaceuticals can be, for example, oral preparations (capsules, tablets, chewable tablets, powders, granules, lozenges, liquids, syrups, etc.).

[0060] The composition of the present invention can be a preparation with, for example, the daily dose or the dose per administration shown below as one unit.

[0061] (Dosage) The daily dose of the composition of the present invention can be appropriately determined according to the use, the state of the individual, body weight, sex, age, the activity of the active ingredient, the route of ingestion or administration, the ingestion or administration schedule, the dosage form, or other factors. Although not particularly limited, for each use, the dosage within the range exemplified below can be set independently.

[0062] In this specification, "dosage" represents the amount of the composition administered or ingested by an individual.

[0063] The daily dose of the composition of the present invention, in terms of the total amount of milk-derived peptides per 1 kg of the subject's body weight, can be, for example, 0.00001 g or more, 0.00002 g or more, 0.00005 g or more, 0.0001 g or more, 0.0002 g or more, 0.0005 g or more, or 0.001 g or more, and can be, for example, 5 g or less, 2 g or less, 1 g or less, 0.5 g or less, 0.25 g or less, 0.12 g or less, 0.06 g or less, 0.02 g or less, or 0.01 g or less. The daily dose of the composition of the present invention, in terms of the total amount of milk-derived peptides per 1 kg of the subject's body weight, can be, for example, 0.00001 g to 5 g, 0.00002 g to 2 g, 0.00005 g to 1 g, 0.0001 g to 0.5 g, 0.0002 g to 0.25 g, 0.0005 g to 0.12 g, or 0.001 g to 0.06 g.

[0064] The daily dosage of the composition of the present invention, when converted to the amount of any one of the above 20 specific peptides per 1 kg of the subject's body weight, can be, for example, 0.000000001 g or more, 0.000000002 g or more, 0.000000005 g or more, 0.00000001 g or more, 0.00000002 g or more, 0.00000005 g or more, or 0.0000001 g or more, and can be, for example, 0.005 g or less, 0.002 g or less, 0.001 g or less, 0.0005 g or less, 0.00025 g or less, 0.00012 g or less, 0.00006 g or less, 0.00002 g or less, or 0.00001 g or less. The daily dosage of the composition of the present invention, when converted to the amount of any one of the above 20 specific peptides per 1 kg of the subject's body weight, can be, for example, 0.000000001 g to 0.005 g, 0.000000002 g to 0.002 g, 0.000000005 g to 0.001 g, 0.00000001 g to 0.0005 g, 0.00000002 g to 0.00025 g, 0.00000005 g to 0.00012 g, or 0.0000001 g to 0.00006 g.

[0065] When the subject is an adult human, in one embodiment, the daily dosage of the composition of the present invention per individual can be changed depending on the attributes of the population such as country, race, gender, etc. In one embodiment, the daily dosage is calculated by selecting the closest value from 50 kg, 60 kg, 70 kg, 80 kg, or 90 kg as the average weight of adult humans in that population and multiplying that number by the dosage per 1 kg of body weight. For example, when targeting Japanese people, the dosage per adult human individual can be calculated by multiplying the dosage per 1 kg of body weight by 60.

[0066] When the subject is a minor such as an infant, elementary school student, or junior high school student, in one embodiment, the daily dosage of the composition of the present invention per individual can be calculated, for example, by multiplying the dosage per 1 kg by an average weight of 30 to 60 kg, or by multiplying by the following average weights according to age: Infant (4 - 6 years old): 16 - 20 kg Elementary school students (7 - 12 years old): 22 - 40 kg Elementary school students to junior high school students (11 - 15 years old), or junior high school students (12 - 15 years old): 40 - 60 kg.

[0067] The frequency of intake or administration of the composition of the present invention can be appropriately determined according to the individual's condition, body weight, gender, age, activity of the material, intake or administration route, dosage, formulation form, or other factors. The frequency of intake or administration of the composition of the present invention can be, for example, once per month, once per week, once every three days, 1 - 6 times a day, 1 - 3 times a day, 1 - 2 times a day, or any period and interval.

Examples

[0068] Next, the present invention will be specifically described by way of examples and test examples, but the present invention is not limited to the following examples and test examples.

[0069] [Test Example 1. Confirmation test of hematopoietic promoting effect using zebrafish (1)] In this test example, zebrafish was used to test whether milk-derived peptides have a hematopoietic promoting effect.

[0070] (Milk-derived peptide) The milk-derived peptide used in this test was prepared by the following method. (1) A reaction solution containing milk-derived protein, neutral protease, and neutral peptidase was adjusted to pH 5 - 8.5, and a hydrolysis reaction was carried out. (2) The reaction solution was heated to inactivate the enzyme. (3) Activated carbon was added to the solution, diatomaceous earth filtration was carried out, followed by concentration under reduced pressure, and then sterilization treatment and spray drying were carried out.

[0071] After excluding the fraction with a molecular weight of 8000 or more from the obtained milk-derived peptide using an ultrafiltration membrane, it was loaded onto a Sephadex G-25 column (φ2.6 × 100 cm), fractionated into 10 mL fractions each, and the molecular weight distribution was examined. The molecular weight distribution of the obtained milk-derived peptide is shown in Table 2.

[0072]

Table 2

[0073] The peptides contained in the fraction of milk-derived peptides with a molecular weight of 2000 or less were fractionated and sequenced using a protein sequencer (manufactured by Applied Biosystem). As a result, the milk-derived peptides contained peptides consisting of the amino acid sequences of KHP (SEQ ID NO: 1), VRY (SEQ ID NO: 2), DIK (SEQ ID NO: 3), KEK (SEQ ID NO: 4), IKHQ (SEQ ID NO: 5), KIHP (SEQ ID NO: 6), SRYP (SEQ ID NO: 7), RYPS (SEQ ID NO: 8), KYIP (SEQ ID NO: 9), IHPF (SEQ ID NO: 10), MKPW (SEQ ID NO: 11), HQPHQ (SEQ ID NO: 12), VEQKH (SEQ ID NO: 13), KDERF (SEQ ID NO: 14), VDDKHY (SEQ ID NO: 15), DDKHYQ (SEQ ID NO: 16), KYKVPQ (SEQ ID NO: 17), VDDKHYQ (SEQ ID NO: 18), VDDKHYQK (SEQ ID NO: 19), and HKEMPFPKY (SEQ ID NO: 20). The total content of these 20 types of peptides in the obtained milk-derived peptides was slightly different depending on the production lot, but was in the range of 0.1 to 1% by mass.

[0074] (Administration to Zebrafish and Measurement) (1) Adult zebrafish aged 6 to 12 months and weighing about 0.3 to 0.5 g were selected and anesthetized in a 500 ppm solution of 2-phenoxyethanol (2-PE) (manufactured by FUJIFILM Wako Pure Chemical Corporation) at -1 week (1 week before the start of administration). The immobilized adult fish were scooped out, placed on a paper towel, and 5 μL of blood was collected from the dorsal artery directly below the body axis using a glass capillary, and the hemoglobin (Hb) value was measured using a hemoglobin assay kit (manufactured by FUJIFILM Wako Pure Chemical Corporation) (normal Hb). The actual blood collection method was based on the inventors' previous academic paper (Zang L. et al., Zebrafish, 2013, Vol.10, Issue 3, pp.425-432). (2) The collected zebrafish were returned to the aquarium system and normally reared. One week later (0 week), 3 μL of blood was collected again, and Hb was measured (Hb at anemia). (3) The collected adult fish were divided into a control group and a milk-derived peptide administration group, and fed three times a day. The control group was administered only gluten, and the milk-derived peptide administration group was administered a gluten diet containing 2.5% by mass of milk-derived peptide. The gluten diet containing milk-derived peptide was prepared based on the inventors' previous academic paper (Zang et al., Zebrafish, 2011, Vol.8, Issue 4, pp.203-210). The dosage of the feed was 30 - 50 μg / g body weight / day. One week after administration, the zebrafish in both groups were euthanized by placing them in ice water after blood collection, and the abdomen was opened to collect the kidneys, hearts, and livers. (4) Total RNA was extracted from each organ using Trizol (manufactured by Thermo Fisher Scientific) and QIAGEN RNeasy Mini-prep Kit (manufactured by Qiagen), and cDNA was synthesized from 500 ng of total RNA using ReverTra Ace qPCR RT Kit (manufactured by Toyobo). Next, qPCR was performed using Power SYBR Green Master Mix (manufactured by Thermo Fisher Scientific) and ABI Stepone Plus Real-Time PCR System (manufactured by Thermo Fisher Scientific). GAPDH was used as the endogenous control gene, and the relative mRNA expression level was analyzed by the ΔΔCt method.

[0075] (Results) Taking the start of administration of the feed containing milk-derived peptide as 0 week, the hemoglobin amount in the blood of each group and the EPO gene expression level in each tissue one week before administration (-1 week) and one week after the end of administration (1 week) are shown in FIGS. 1A and 1B, respectively. In the milk-derived peptide administration group, compared with the control group, the hemoglobin amount in the blood and the EPO gene expression level in the kidneys increased significantly after feed administration.

[0076] [Test Example 2. Confirmation Test of Hematopoietic Promotion Effect Using Zebrafish (2)] In this experiment, a test was conducted to confirm the hematopoietic promoting effect when milk-derived peptides were administered to bled zebrafish over a longer period. The same feed containing milk-derived peptides as in Test Example 1 was used. qPCR was performed on the heart one week after administration in the same manner as in Test Example 1.

[0077] (Administration to Zebrafish and Measurement) The administration test to zebrafish and the measurement of each outcome were performed according to the following procedure. A schematic diagram of the test procedure is shown in Figure 2. The test was carried out in the same procedure as in Test Example 1, except that the administration was carried out for up to 3 weeks and blood and each tissue were collected from separate individuals every week. The same milk-derived peptides as in Test Example 1 were used.

[0078] (Results) The measurement results of the hemoglobin amount in the blood are shown in Figure 3. The milk-derived peptide administration group showed a significantly higher hemoglobin amount in the blood even 3 weeks after administration compared to the control group.

[0079] The results of qPCR are shown in Figure 4. The expression of the EPO gene (epoa, transcript variant 1) was significantly promoted by the administration of milk-derived peptides. The expression of the IGF1 gene was also promoted. In zebrafish, the hif1aa and hif1bb genes exist as homologs of the hypoxia-inducible factor-1α subunit (HIF1A) gene, and the expression of both was promoted by the administration of milk-derived peptides.

[0080] When the blood volume in the body decreases, it is known that the expression of insulin-like growth factor-1 (IGF-1) gene and low HIF1A gene increases due to hypoxia stimulation. Due to the expression of IGF1 protein, the expression of HIF1A gene increases from the IGF-1 receptor through the Akt and ERK signaling systems. And the expression of EPO is promoted by the HIF-1 protein which is a transcription factor. According to the above results, it is speculated that the milk-derived peptide of the present invention promoted the expression of the EPO gene through HIF-1 by promoting the expression of the IGF-1 gene or the IGF-1 and HIF-1 genes. And it is speculated that the milk-derived peptide of the present invention finally promotes the differentiation of erythrocytes and increases the amount of hemoglobin by improving the expression of EPO.

Claims

1. A composition for improving the amount of hemoglobin in blood, containing at least one selected from the group consisting of milk-derived peptides, derivatives thereof, and salts thereof as an active ingredient.

2. A composition for improving the number of red blood cells in blood, containing at least one selected from the group consisting of milk-derived peptides, derivatives thereof, and salts thereof as an active ingredient.

3. A composition for promoting the production of erythropoietin, containing at least one selected from the group consisting of milk-derived peptides, derivatives thereof, and salts thereof as an active ingredient.

4. A composition for preventing or improving anemia, containing at least one selected from the group consisting of milk-derived peptides, derivatives thereof, and salts thereof as an active ingredient.

5. The composition according to any one of Claims 1 to 4, for improving any one of the following (a) to (d): (a) Poor complexion or pale palpebral conjunctiva, (b) Headache, dizziness, or lightheadedness, (c) Sense of fatigue, lassitude, or weakness, (d) Shortness of breath or increased heart rate.

6. The composition according to any one of Claims 1 to 4, which is administered or ingested to any of the following individuals: (a) Individuals with iron deficiency without anemia (IDNA), (b) Individuals who perform high-load exercise daily, (c) Women.

Images