Bone formation accelerating composition

Abstract

To provide new means for accelerating bone formation including bone growth.SOLUTION: Provided are the following items, each containing as active principle at least one kind selected from a group consisting of milk-derived peptide, a derivative thereof, and salt thereof: a bone growth accelerating and / or improving composition; a bone formation improving composition; a body length augmentation accelerating composition; a physical constitution improving and / or growth improving composition; a bone strengthening composition; a cartilage quantity improving or cartilage function improving composition; and an insulin-like growth factor (IGF-1) producing accelerating composition.SELECTED DRAWING: Figure 3

Description

Technical Field

[0001] The present invention relates to a composition for promoting bone formation including bone growth.

Background Art

[0002] In growing individuals, bone is formed while growing mainly by endochondral ossification. In endochondral ossification, a quiescent chondrocyte layer that serves as a source of chondrocytes that form cartilage, which is the basis of bone formation, a proliferating chondrocyte layer that increases chondrocytes, and a hypertrophic chondrocyte layer that hypertrophies and exhibits apoptosis are stratified cell groups that form growth cartilage (also called the "growth plate"). As long as cell supply from the quiescent chondrocyte layer to the proliferating chondrocyte layer continues, bone growth occurs. Toward the end of the growth period, the supply of chondrocytes decreases, the chondrocytes hypertrophy and undergo apoptosis, and further ossification occurs, resulting in the cessation of bone growth (see Non-Patent Document 1). Thus, in the growth period, the formation of cartilage (growth plate), which is the basis of bone, is important. If ossification is simply promoted and ossification proceeds in a state where cartilage formation is insufficient, it may rather inhibit bone growth.

[0003] Also, when a gap occurs in the bone due to a fracture, it is repaired by bone formation by endochondral ossification similar to the above.

[0004] On the other hand, in the growth and mature periods, bone resorption by osteoclasts and bone formation by osteoblasts are constantly carried out, and bone is remodeled in about 2 to 5 months. When bone resorption becomes significant compared to bone formation, bone mass decreases, leading to an increase in the risk of osteoporosis and fractures. After passing through the growth period with significant bone growth, it is important to maintain the balance between bone resorption and bone formation in order to maintain bone mass.

[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

[0006] [Patent Document 1] JP 2009-263255 A [Non-patent literature]

[0007] [Non-Patent Document 1] Journal of Biomechanisms, 2008, Vol. 32, No. 2, pp. 57-60 Summary of the Invention [Problem to be solved by the invention]

[0008] To achieve healthy bone formation during the growth stage, it is important to maintain a healthy timing and balance between cartilage formation and ossification. Therefore, a means other than those used to maintain the balance between bone resorption and bone formation in mature individuals in which bone elongation has ceased is needed. On the other hand, even in mature individuals, promotion of bone formation through ossification of cartilage may be required under circumstances in which bone repair is required.

[0009] In addition, as for the reduction in bone mass during maturity, measures such as increasing calcium intake are known as a means for suppressing bone resorption, but measures for promoting bone formation are limited.

[0010] Therefore, an object of the present invention is to provide a new means for promoting bone formation, including bone growth. [Means for solving the problem]

[0011] As a result of intensive research, the present inventors have found that a composition containing a specific milk-derived peptide promotes bone growth and bone formation, as well as cartilage formation. They have also found that a composition containing a specific milk-derived peptide promotes the production of insulin-like growth factor (IGF-1). Based on the above, the present inventors have completed the present invention.

[0012] That is, the present invention includes, but is not limited to, the following aspects. [1] A composition for promoting and / or improving bone growth, containing at least one selected from the group consisting of milk-derived peptides, their derivatives, and their salts as an active ingredient. [2] A composition for improving bone formation, 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 body length increase, 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 improving physique and / or growth, containing at least one selected from the group consisting of milk-derived peptides, their derivatives, and their salts as an active ingredient. [5] A composition for improving cartilage mass or improving cartilage function, containing at least one selected from the group consisting of milk-derived peptides, their derivatives, and their salts as an active ingredient. [6] A composition for strengthening bone, containing at least one selected from the group consisting of milk-derived peptides, their derivatives, and their salts as an active ingredient. [7] A composition for promoting the production of insulin-like growth factor (IGF-1), containing at least one selected from the group consisting of milk-derived peptides, their derivatives, and their salts as an active ingredient. [8] The composition according to any one of [1] to [7], comprising a peptide consisting of at least one 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).

Advantages of the Invention

[0013] The composition of the present invention can be administered to a subject to improve bone formation including bone growth, promote an increase in body length, and improve physique and growth.

[0014] In addition, the composition of the present invention can improve the amount of cartilage by administering it to a subject.

Brief Description of the Drawings

[0015]

Figure 1

Figure 2

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Figure 6

Figure 7

Mode for Carrying Out the Invention

[0016] 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.

[0017] (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, for example, by any of the following methods (i) to (iii).

[0018] <Aspect (i) Preparation by hydrolysis of milk-derived protein> (i) In one embodiment, the milk-derived peptide is prepared by hydrolysis of milk-derived protein. This preparation method is particularly preferred because of its low cost and ease of large-scale production.

[0019] 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.

[0020] Operations such as hydrolysis of milk-derived proteins can be carried out, for example, according to the method described in International Publication No. WO89 / 06970. Hydrolysis can be carried out using, for example, 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.

[0021] 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.

[0022] 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).

[0023] From the hydrolyzate of milk-derived proteins, water-insoluble fractions (such as insoluble proteins containing inactivated enzymes) may be removed as appropriate by known methods such as filtration. For example, when the enzyme is inactivated by heating, it is preferable to remove the water-insoluble fraction 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.

[0024] <Embodiment (ii) Preparation by Chemical Synthesis> (ii) In another embodiment, the milk-derived peptide is chemically synthesized from among the peptides obtained in embodiment (i) that have the action according to the present invention by known techniques (for example, solid-phase peptide synthesis using protecting groups such as tert-butyloxycarbonyl group, 9-fluorenylmethyloxycarbonyl group, liquid-phase method, etc.).

[0025] <Aspect (iii) Preparation by biosynthetic methods> (iii) In yet another embodiment, the milk-derived peptide is produced by a biosynthetic method involving 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 the 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.

[0026] 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 methods different from them 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 appropriately known methods.

[0027] 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 molecular weight for the preparation containing the milk-derived peptide (especially the preparation of aspect (i)). For molecular weight fractionation, for example, ultrafiltration or gel filtration chromatography can be used.

[0028] The weight average molecular weight of the milk-derived peptide obtained by 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 effects of the present invention and being highly water-soluble.

[0029] In the present specification, the weight average molecular weight of the peptide is measured by the gel permeation chromatography (GPC) method.

[0030] In the method for producing milk-derived peptides, depending on the production method, form, use, etc., it may further include a concentration step, a drying step, a sterilization step, a packaging step, etc. 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.

[0031] 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.

[0032] 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 parts by mass, and particularly more preferably 0.4 - 0.8 parts by mass.

[0033] 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 may 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.

[0034] 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.

[0035] In one embodiment, the composition of the present invention contains milk-derived peptides, their derivatives and their salts, 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".

[0036] 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, even more preferably 0.2% by mass or more, based on 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.

[0037] The preferred range of the content of other polypeptides relative to 1 part by mass of KHP (SEQ ID NO: 1) in milk-derived peptides 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 milk-derived peptides is the combination described in the "preferred range", "more preferred range", "even more preferred range" or "even more preferably range" in Table 1.

[0038]

Table 1

[0039] In the present specification, the salts of milk-derived peptides are not particularly limited as long as they are pharmacologically or physiologically acceptable salts, and specifically include 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.

[0040] In this specification, a "derivative" of a milk-derived peptide refers to a chemically modified product obtained by subjecting a milk-derived peptide as a raw material to 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 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.).

[0041] (Other components) Depending on the use, form, etc. of the composition of the present invention, in addition to the above milk-derived peptides, the composition may further contain other components such as a base, a carrier, an additive, bone-related components [such as calcium, phosphorus, vitamin D (vitamin D2, vitamin D3, etc.), other known bone resorption inhibitory components or bone formation promoting components, etc.], cartilage-related components [such as glucosamine, its derivatives or their salts, chondroitin sulfate, cartilage-derived proteoglycan (such as from salmon bone), native type II collagen, collagen peptide, chicken foot enzyme-digested peptide, elastin peptide (such as from bonito), curcuminoids, rhamdan, quercetin glycoside, etc.].

[0042] Examples of bone resorption inhibitory components or bone formation promoting components include, but are not limited to, Bonepep (registered trademark, Pharmafuz Co., Ltd.), β-cryptoxanthin, β-carotene, zeaxanthin, capsanthin, isoflavone (such as soy isoflavone), calcium maltobionate, MBP (milk basic protein), Bacillus subtilis strain C-3102, Bifidobacterium bifidum strain BB536, etc.

[0043] (Use) As shown in the following examples, the composition of the present invention has an effect of promoting bone growth and an effect of promoting body length increase. Therefore, the composition of the present invention is suitable for uses such as improving bone growth including promoting bone growth or promoting body length increase. As described above, for bone growth, formation of cartilage as a scaffold is essential. Therefore, although the bone growth promoting effect and body length increase promoting effect of the present invention may be due in part to promotion of cartilage formation by milk-derived peptides, the present invention is not limited by such a mechanism.

[0044] In the present specification, "improvement of bone growth" more specifically includes maintaining the bone growth of an individual in a healthy state, approaching the bone growth of a healthy individual, or promoting the bone growth of an individual. Maintaining the bone growth of an individual in a healthy state includes, for example, maintaining the bone mass per body length at the level of a healthy individual. Approaching the bone growth of a healthy individual includes, for example, approaching the bone mass per body length to the level of a healthy individual.

[0045] As shown in the following examples, the composition of the present invention has an effect of promoting an increase in bone mass relative to body length, and thus is also suitable for uses such as improving physique or growth.

[0046] As shown in the examples, the composition of the present invention promotes the expression of insulin-like growth factor-1 (IGF-1) gene. Therefore, the composition of the present invention has an effect of promoting IGF-1 gene expression or promoting IGF-1 production.

[0047] IGF-1 is known to promote the development, growth, and maturation of a living body. Therefore, the increase in body length, promotion of bone growth, improvement of physique, or improvement of growth of the composition of the present invention may be due in part to promotion of IGF-1 production, but the present invention is not limited by such a mechanism.

[0048] As used herein, "physical improvement" more specifically includes maintaining the healthy development of an individual's physique, approaching the physique of a healthy individual, or promoting the development of an individual's physique. Maintaining the healthy development of an individual's physique includes, for example, maintaining the bone mass per body length at the level of a healthy individual. Approaching the physique of a healthy individual includes, for example, approaching the bone mass per body length to the level of a healthy individual.

[0049] As used herein, "growth improvement" more specifically includes maintaining the healthy growth of an individual, approaching the growth of a healthy individual, or promoting the growth of an individual. Maintaining the healthy growth of an individual includes, for example, approaching the growth of an individual to the average body length based on the individual's age and gender. Body length is height in the case of humans.

[0050] As shown in the following examples, the composition of the present invention promotes bone growth while also promoting the ossification from cartilage to calcified bone, and thus promotes bone formation. Therefore, the composition of the present invention is suitable for use in improving bone formation. By improving bone formation, the bone is strengthened, so the composition of the present invention is also suitable for use in bone strengthening. The composition of the present invention is suitable for use in bone formation during the growth period in that it is less likely to cause inhibition of bone growth due to excessive ossification of cartilage during the growth period.

[0051] As used herein, "improvement of bone formation" more specifically includes maintaining the health of an individual's bones, approaching the bone formation of a healthy individual, or promoting the bone formation of an individual. Maintaining the health of an individual's bones includes, for example, maintaining the bone mass per body length at the level of a healthy individual. Approaching the bone formation of a healthy individual includes, for example, approaching the bone mass per body length to the level of a healthy individual.

[0052] As shown in the following examples, the composition of the present invention has an effect of promoting cartilage formation, and thus is suitable for use in improving cartilage mass. Through the improvement of cartilage mass, the composition of the present invention is also suitable for use in improving cartilage function. The site of cartilage to which the composition of the present invention is applied is not particularly limited, and examples include cartilage formed during bone growth (e.g., the growth plate of bone), and cartilage of joints such as the knee, shoulder, elbow, cervical vertebra, spine, lumbar vertebra, and hip joint. Among them, cartilage formed during bone growth is preferred.

[0053] In this specification, "improvement of cartilage mass" more specifically includes maintaining the amount of cartilage of an individual or a specific site of an individual at a healthy level, promoting an increase in cartilage mass, suppressing, delaying or preventing a decrease in cartilage mass, or reducing the risk of a decrease in cartilage mass.

[0054] Improvement of cartilage function includes, for example, maintaining and promoting the formation of growth plates in endochondral ossification and improving bone growth.

[0055] The composition of the present invention may be used for therapeutic purposes or non-therapeutic purposes. In this specification, "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 doctor's instruction (for example, uses related to health maintenance or promotion, or beauty).

[0056] (Applicable Subjects) The composition of the present invention can be used for vertebrates such as mammals (human or non-human), birds (budgerigar, parrot, pigeon, finch, owl, nightingale, etc.), reptiles (snake, turtle, lizard, etc.), amphibians (frog, newt, etc.), and fish. In one embodiment, the composition of the present invention is used for human or non-human mammals, preferably humans.

[0057] Non-human mammals include, for example, 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, hedgehogs, monkeys, chimpanzees, rabbits, degus, squirrels, martens, rats, voles, chinchillas, ferrets, camels, wild boars, giraffes, deer, bison, whales, dolphins, lions, tigers, wolves, weasels, bears, seals, etc., and more preferably dogs or cats.

[0058] Fish include cultured 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.

[0059] In one embodiment, the composition of the present invention is applied to individuals in the growth period. Individuals in the growth period are preferred because the effects corresponding to the above respective uses are particularly remarkable.

[0060] Human individuals in the growth period are, 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 human individuals in the growth period include, but are not limited to, 0 - 18 years old, 3 - 16 years old, 6 - 14 years old, or 6 - 12 years old.

[0061] In another embodiment, the composition of the present invention is applied to individuals in the mature period after the growth period. The composition of the present invention may be applied for any of the above uses to individuals in the mature period (particularly humans).

[0062] Human individuals in the mature period 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 period can be, for example, 20 - 50 years old, 25 - 45 years old, or 25 - 40 years old, but are not limited thereto.

[0063] The composition of the present invention is applied, for example, for the use of promoting bone formation in individuals in the mature stage. In this embodiment, among individuals in the mature stage, it is more preferably applied to individuals who need to promote bone growth or bone formation due to fractures or the like, or individuals who have the possibility or risk of bone mass reduction due to bone resorption (for example, individuals with low body weight, lack of exercise, advanced age, genetic factors, low calcium intake, etc.).

[0064] The composition of the present invention is applied, for example, for the use of suppressing cartilage reduction, promoting cartilage formation, or improving cartilage function in individuals in the growth stage or mature stage. In this embodiment, it is more preferably applied to individuals who have joint abnormalities or the like accompanied by cartilage reduction in joints or the like, or individuals who have the possibility or risk of cartilage reduction (for example, humans aged 60 or above, 70 or above, or 80 or above, individuals who have continuously engaged in exercises that load the joints, etc.).

[0065] The gender of the subject to which the composition of the present invention is applied is not particularly limited. In the use of promoting bone formation in individuals, in humans, women (for example, 40 years old or above, preferably 50 years old or above) are known to have the possibility or risk of bone mass reduction due to bone resorption, and are preferred subjects.

[0066] (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, and foods for specified health uses, etc.), pharmaceuticals, quasi-drugs (drinkable agents, 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 they are.

[0067] In one embodiment, the composition of the present invention has a product label or attached document of a pharmaceutical or the like that explicitly or implicitly indicates any of the above uses or the actions related thereto. Examples of such compositions include pharmaceuticals, supplements, foods with functional claims, foods for patients, foods for specified health uses, or nutritional functional foods, etc.

[0068] Examples of expressions that explicitly or implicitly indicate the use for improving bone growth, promoting body length increase, or improving physique include expressions that explicitly or implicitly indicate the improvement of bone or body growth, such as "promote the development of a healthy body", "assist in healthy (bone) growth", "maintain healthy (bone) growth", "support (bone) growth", "support (bone) development", etc., and expressions that recommend to individuals in the growth period, which is the target suitable for the use of such improvements, such as "support the growth period", "want to extend the potential of the growth period", "for the nutrition in the growth period", etc.

[0069] Examples of expressions that explicitly or implicitly indicate the use for promoting bone formation or strengthening bone include, in addition to expressions indicating an increase in bone mass or bone density such as "increase bone density", expressions that explicitly or implicitly indicate maintaining a healthy state with high bone density, such as "for maintaining strong bones", "maintaining bone density".

[0070] Examples of expressions that explicitly or implicitly indicate the use for suppressing cartilage reduction or promoting cartilage formation include, in addition to expressions indicating suppressing cartilage reduction such as "suppress the decomposition of cartilage components" or promoting cartilage formation, expressions that explicitly or implicitly indicate maintaining a healthy state with high bone density, such as "maintaining cartilage".

[0071] When the composition of the present invention is a food or drink, its form can be, for example, liquid drinks such as soups, juices, fruit juice drinks, milk, milk drinks, whey drinks, lactic acid bacteria drinks, tea drinks, alcoholic drinks, 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.).

[0072] The pharmaceutical can be, for example, an oral preparation (capsules, tablets, chewable tablets, powders, granules, lozenges, liquids, syrups, etc.).

[0073] The composition of the present invention can be, for example, a preparation with the daily dosage or the dosage per administration shown below as one unit.

[0074] (Dosage) The daily dosage of the composition of the present invention can be appropriately determined according to the use, the state of the individual, body weight, sex, age, activity of the active ingredient, intake or administration route, intake or administration schedule, dosage form or other factors. Although not particularly limited, for each use, the dosages within the ranges exemplified below can be independently set.

[0075] In this specification, "dosage" refers to the amount of the composition administered or ingested by an individual.

[0076] The daily dosage of the composition of the present invention, when converted to 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 dosage of the composition of the present invention, when converted to 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.

[0077] 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.

[0078] 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 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.

[0079] 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 the following average weights by 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.

[0080] 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 a month, once a 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.

Example

[0081] 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.

[0082] [Test Example 1. Confirmation test of bone formation promoting effect using zebrafish] Using zebrafish, the bone formation promoting effect of milk-derived peptides was tested. When zebrafish grow from larvae to adults, cartilage is formed in the neural spines and caudal fins and eventually ossifies to promote bone growth. The bone growth of zebrafish occurs in a manner common to humans and other vertebrates and is used as a model for bone growth in vertebrates including humans (see, for example, Carnovali M et al., Marine Drugs, 2022, Vol.20, Issue 2, 135; URL:https: / / www.mdpi.com / 1660 - 3397 / 20 / 2 / 135).

[0083] (Milk-derived peptides) The milk-derived peptides used in this test were 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 subjected to a hydrolysis reaction. (2) The reaction solution was heated to inactivate the enzyme. (3) Activated carbon was added to the solution, followed by diatomaceous earth filtration, then vacuum concentration, and finally sterilization treatment and spray drying.

[0084] The obtained milk-derived peptides were subjected to ultrafiltration through a membrane to exclude fractions with a molecular weight of 8000 or more, and then loaded onto a Sephadex G-25 column (φ2.6×100 cm), fractionated into 10 mL fractions each to examine the molecular weight distribution. The molecular weight distribution of the obtained milk-derived peptides is shown in Table 2.

[0085]

Table 2

[0086] The peptides contained in the fraction of the 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 varied slightly depending on the production lot, but was in the range of 0.1 to 1% by mass.

[0087] (Administration to zebrafish and measurement) (1) Zebrafish (wild-type AB strain) fertilized eggs were placed in rearing water (0.3× Danieau’s solution; [17.4 mM NaCl, 0.21 mM KCl, 0.12 mM MgSO4, 0.18 mM Ca(NO3)2, 1.5 mM HEPES (pH 7.2)]), and rearing was started under an environment of 28°C. (2) Two days after hatching, the juvenile fish were divided into four groups (100 individuals per group) and administered milk-derived peptides by exposing them to 200 mL of rearing water containing 0, 3.3, 10, or 33 μg / mL of milk-derived peptides. 0 μg / mL was the control group. The rearing water was exchanged by half (100 mL) every day, and a continuous test was conducted for 21 days. Separately, as feed, Artemia nauplii (Kyorin Co., Ltd.) was fed to each group in the same amount from the 4th to the 6th day, and Gemma Micro 75 (SKRETTING Co., Ltd.) was fed to each group in the same amount after the 7th day. (3) After the administration test was completed, the zebrafish were immersed and fixed in 4% paraformaldehyde phosphate buffer for 2 hours or more, and double staining with alizarin red (bone) and alcian blue (cartilage) was performed. Specifically, the method of Walker MB & Kimmel CB (Walker, M.B. & Kimmel, C.B, Biotechnic & Histochemistry, 2007, Vol.82, pp.23-28) was used. After staining, the zebrafish were dehydrated with glycerol, and color photography was performed with an inverted microscope BZ-X710 (Keyence Corporation), and a whole-body image was obtained using its tiling function. (4) Using "Alcian blue & H" in the Colour Deconvolution function of ImageJ (Fiji distribution, NIH), the regions of alcian blue (cartilage) and alizarin red (bone) were extracted. The staining amount in the region was quantified and used as the bone and cartilage amount. Note that only the whole body for bone and the caudal fin part for cartilage were quantified.

[0088] (Results) The microscopic images of each group under bright field are shown in Fig. 2. The higher the dosage of the milk-derived peptide, the more significantly the ossification of the neural spine and caudal fin was promoted. Since the space between the vertebrae decreased with the increase in the dosage of the milk-derived peptide, it was confirmed that the ossification and bone growth of the vertebrae were also significantly promoted. In addition, it was confirmed that the higher the amount of the milk-derived peptide, the more the formation of cartilage in the caudal fin and anal fin was promoted.

[0089] The body length and the number of ossified neural spines of each group are shown in Fig. 3. In addition, the number of individuals with ossified caudal fins is shown in Fig. 4. The number of ossified neural spines and the number of individuals with ossified caudal fins were significantly increased in the group administered with the milk-derived peptide compared to the control. It was also revealed that even the diet containing 3 μg / mL of the milk-derived peptide sufficiently promoted ossification. Therefore, it was clarified that the milk-derived peptide promotes bone formation, particularly bone growth during the growth period. Furthermore, in the diet containing the milk-derived peptide, the body length was also significantly increased compared to the control. Therefore, it was clarified that the milk-derived peptide promotes the increase in body length without hindering bone growth due to excessive ossification or the like during bone growth.

[0090] The ossified area (pixels) and the ossified area per body length (pixels / mm) obtained by the analysis using ImageJ are shown in Fig. 5. Even when the ossified area was divided by the body length to compare the ossified area per body length, the diet containing the milk-derived peptide showed a significantly larger value compared to the control. The larger the bone mass per body length, the stronger and more robust the physique. Therefore, it was found that the milk-derived peptide is effective not only in increasing the body length but also in improving the physique.

[0091] The area of cartilage (pixels) in the caudal fin region obtained by the analysis using ImageJ is shown in Fig. 6. In the diet containing 3 μg / mL of the milk-derived peptide, the area of cartilage increased compared to the control. Therefore, it was found that the milk-derived peptide is also effective in promoting cartilage formation. When the amount of the milk-derived peptide was further increased, the ossification of cartilage also progressed, resulting in a slight decrease in the area value.

[0092] [Test Example 2. Test for Confirming the Effect of Promoting IGF-1 Production Using Zebrafish] In this test, a test was conducted to confirm the IGF-1 production effect by administering milk-derived peptides to zebrafish with bloodletting. The same feed containing milk-derived peptides as in Test Example 1 was used.

[0093] (Administration to Zebrafish and Measurement) The administration test to zebrafish and the measurement of each outcome were performed according to the following procedure. The same milk-derived peptides as in Test Example 1 were used. (1) Adult zebrafish aged 6 - 12 months and weighing approximately 0.3 - 0.5 g were selected and anesthetized in a 500 ppm 2-phenoxyethanol (2-PE) (manufactured by FUJIFILM Wako Pure Chemical Corporation) solution at -1 week (1 week before the start of administration). The adult fish whose movement had stopped were scooped out, placed on a paper towel, and 5 μL of blood was collected from the dorsal aorta directly below the body axis using a glass capillary. 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' past academic papers (Zang L. et al., Zebrafish, 2013, Vol.10, Issue 3, pp.425 - 432). (2) The blood-sampled zebrafish were returned to the aquarium system and normally reared. After 1 week (0 week), 3 μL of blood was collected again and Hb was measured (Hb during anemia). (3) The blood-sampled 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 peptides. The gluten diet containing milk-derived peptides was prepared based on the inventors' past academic papers (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. After 1 week of administration, the zebrafish in both groups were bled, then placed in ice water to be euthanized, and the abdomen was opened to collect the kidneys, hearts, and livers. (4) Total RNA was extracted from each organ using Trizol (Thermo Fisher Scientific) and the QIAGEN RNeasy Mini-prep Kit (Qiagen), and cDNA was synthesized from 500 ng of total RNA using the ReverTra Ace qPCR RT Kit (Toyobo). Next, qPCR was performed using the Power SYBR Green Master Mix (Thermo Fisher Scientific) and the ABI Stepone Plus Real-Time PCR System (Thermo Fisher Scientific). GAPDH was used as the endogenous control gene, and the relative mRNA expression level was analyzed by the ΔΔCt method.

[0094] (Results) The results of qPCR are shown in Fig. 7. The administration of milk-derived peptides significantly promoted the expression of the IGF1 gene.

Claims

1. A composition for promoting and / or improving bone growth, comprising 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 bone formation, comprising 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 body length increase, comprising 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 improving physique and / or growth, comprising at least one selected from the group consisting of milk-derived peptides, derivatives thereof, and salts thereof as an active ingredient.

5. A composition for improving cartilage mass or improving cartilage function, comprising at least one selected from the group consisting of milk-derived peptides, derivatives thereof, and salts thereof as an active ingredient.

6. A composition for strengthening bone, comprising at least one selected from the group consisting of milk-derived peptides, derivatives thereof, and salts thereof as an active ingredient.

7. A composition for promoting the production of insulin-like growth factor (IGF-1), comprising at least one selected from the group consisting of milk-derived peptides, derivatives thereof, and salts thereof as an active ingredient.

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