JP7168477B2 - Food composition and osteoblast differentiation promoter - Google Patents

Description

TECHNICAL FIELD The present invention relates to a food composition having an excellent effect of promoting differentiation into osteoblasts. Furthermore, the present invention relates to an osteoblastic differentiation promoter, an osteogenesis promoter, and a pharmaceutical composition for treating and/or preventing bone weight loss diseases.

Bone is constantly reborn while maintaining dynamic homeostasis through a delicate balance between destruction and formation. This remodeling, called bone remodeling, involves osteoclasts resorbing old bone (
destruction), a series of processes begin (absorption period). Next, old osteoclasts are phagocytosed by macrophages, their function is inhibited (reversal phase), and osteoblasts secrete and fill the missing bone matrix with bone matrix (plastic phase). After the bone is supplemented by bone formation by osteoblasts, bone formation is halted (quiescent phase). Thus, the balance between bone destruction and formation is strictly regulated by both osteoblasts responsible for bone formation and osteoclasts responsible for bone resorption. It is known to cause various bone diseases such as osteoporosis.

Currently, bisphosphonate preparations and the like are used as therapeutic agents for osteoporosis. However, there are still unresolved problems in terms of not only therapeutic efficacy but also safety, etc., and there is a demand for the development of further therapeutic drugs that solve these problems. In particular, materials that can be used for both food and medicine are desirable from the viewpoint of daily use.

Glucosamine is present as a constituent of glycosaminoglycans in connective and cartilage tissues and contributes to the strength, flexibility and elasticity of these tissues. For food, it is collected from crabs, shrimps, etc. and used. Glucosamine intake is known to normalize cartilage metabolism and has been used to treat osteoarthritis in humans. In addition, Non-Patent Document 1 reports that glucosamine promotes differentiation of osteoblasts that generate new bones.

Similarly, chondroitin sulfate is known to exist as a constituent of glycosaminoglycan in connective tissue and cartilage tissue, and is collected and used as food from cartilage tissue of sharks and the like. Ingestion of chondroitin sulfate is believed to be effective against osteoarthritis in humans. In addition, Non-Patent Document 2 reports that chondroitin sulfate suppresses differentiation of osteoclasts. Furthermore, Non-Patent Document 3 reports that a combination of chondroitin sulfate and glucosamine suppresses osteoblast resorption of subchondral bone in human osteoarthritis.

Royal jelly is a milk-like substance that is synthesized in the salivary glands of honeybees and secreted from the Mandebler glands in the head of the bee. be. Currently, royal jelly is widely used in health foods, pharmaceuticals, and cosmetics all over the world. Tumor action, wound healing promotion action, autonomic imbalance healing action, anti-stress action, calcium absorption promotion action, blood sugar lowering action, antioxidant action, etc. have been reported. Furthermore, Non-Patent Document 4 confirmed that royal jelly was effective in preventing bone loss in ovariectomized rats, and Non-Patent Document 5 related to bone formation through its action on osteoblasts. It has been reported that royal jelly may be used to prevent osteoporosis, as it has been confirmed to stimulate activity.

M. Igarashi et al., Int J Mol Med, 28(3), 373-9(2011) T. Miyazaki et al., Dental Materials Journal, 29(4), 403-10(2010) S.K. Tat et al., Arthritis Research & Therapy 2007, 9:R117 (doi:10.1186/ar2325) S. Hidaka et al., eCAM, 3(3), 339-348(2006) Y. Narita et al., Biosci. Biotechnol. Biochem., 70(10), 2508-2514(2006)

However, in response to the above report, the inventors evaluated the osteoblast differentiation-promoting action of glucosamine/chondroitin sulfate and royal jelly, and verified the preventive effect against osteoporosis, and found that the effectiveness of each was not high.

Accordingly, an object of the present invention is to provide a food composition having an excellent effect of promoting differentiation into osteoblasts. Another object of the present invention is to provide an agent for promoting differentiation into osteoblasts, an agent for promoting osteogenesis, and a pharmaceutical composition having therapeutic and/or preventive effects on bone weight loss diseases.

The present inventors have found that by using royal jelly in combination with glucosamine and chondroitin sulfate, the effect of promoting differentiation into osteoblasts is obtained by using royal jelly, or glucosamine and chondroitin as described in Non-Patent Documents 3 to 5. It was found that an excellent synergistic effect is exhibited as compared with the case where sulfuric acid is used alone.

The present invention has been completed through further studies based on these findings, and provides the following food composition, osteoblastic differentiation promoter, osteogenesis promoter and pharmaceutical composition. .

Section 1. A food composition comprising royal jelly, glucosamine and chondroitin sulfate.
Section 2. Item 2. The food composition according to item 1, wherein 1 to 20 parts by weight of glucosamine and 0.01 to 10 parts by weight of chondroitin sulfate are contained with respect to 1 part by weight of royal jelly.
Item 3. Item 3. The food composition according to item 1 or item 2, wherein 4 to 20 parts by weight of glucosamine and 0.2 to 10 parts by weight of chondroitin sulfate are contained with respect to 1 part by weight of royal jelly.
Section 4. Item 3. The food composition according to Item 1 or Item 2, wherein 1 to 12 parts by weight of glucosamine and 0.01 to 2 parts by weight of chondroitin sulfate are contained with respect to 1 part by weight of royal jelly.
Item 5. A differentiation promoter to osteoblasts containing royal jelly, glucosamine and chondroitin sulfate.
Item 6. Based on 1 part by weight of royal jelly, 1 to 20 parts by weight of glucosamine (preferably 4 to 20 parts by weight or 1 to 12 parts by weight), 0.01 to 10 parts by weight of chondroitin sulfate (preferably 0.2 to 10 parts by weight or Item 5. The agent for promoting differentiation into osteoblasts according to Item 5, which is included in the product (0.01 to 2 parts by weight).
Item 7. osteogenic agents including royal jelly, glucosamine and chondroitin sulfate;
Item 8. Based on 1 part by weight of royal jelly, 1 to 20 parts by weight of glucosamine (preferably 4 to 20 parts by weight or 1 to 12 parts by weight), 0.01 to 10 parts by weight of chondroitin sulfate (preferably 0.2 to 10 parts by weight or 0.01 to 2 parts by weight), the osteogenesis promoter according to Item 7.
Item 9. A pharmaceutical composition comprising royal jelly, glucosamine and chondroitin sulfate.
Item 10. Based on 1 part by weight of royal jelly, 1 to 20 parts by weight of glucosamine (preferably 4 to 20 parts by weight or 1 to 12 parts by weight), 0.01 to 10 parts by weight of chondroitin sulfate (preferably 0.2 to 10 parts by weight or 0.01 to 2 parts by weight), the pharmaceutical composition according to Item 9.
Item 11. Item 11. The pharmaceutical composition according to Item 9 or 10, which is used for treatment and/or prevention of bone weight loss disease.

The food composition of the present invention has an excellent effect of promoting differentiation into osteoblasts due to the synergistic effect of royal jelly, glucosamine and chondroitin sulfate. Therefore, the combination of royal jelly with glucosamine and chondroitin sulfate is useful as an agent for promoting differentiation into osteoblasts, an agent for promoting osteogenesis, and a component of a pharmaceutical composition (particularly for treating and/or preventing bone weight loss diseases). be.

Moreover, the composition of the present invention is highly safe because it uses royal jelly, glucosamine and chondroitin sulfate, which have been conventionally used as food materials.

It is a graph which shows the result about the differentiation promotion effect|action of an osteoblast. It is a graph which shows the result about the differentiation inhibitory effect|action of an osteoclast.

The present invention will be described in detail below.

The food composition, the agent for promoting differentiation into osteoblasts, the agent for promoting osteogenesis, and the pharmaceutical composition of the present invention are characterized by containing royal jelly, glucosamine and chondroitin sulfate.

Glucosamine in the present invention includes both glucosamine (2-amino-2-deoxyglucose) and its physiologically acceptable salts (eg, hydrochloride, sulfate, phosphate, etc.). Glucosamine is obtained by replacing the hydroxyl group of glucose with an amino group, and is biosynthesized in vivo from glucose and glutamine. Glucosamine is contained in polysaccharides, mucopolysaccharides, glycolipids, glycoproteins, bacterial cell wall peptidoglycans, lipopolysaccharides, etc. of animals, plants and microorganisms. It is distributed in connective tissues such as tendons and ligaments. As glucosamine, chemically synthesized products, commercially available products, and the like can be used.

Chondroitin sulfate in the present invention includes both chondroitin sulfate and physiologically acceptable salts thereof (for example, salts with alkali metals such as potassium and sodium, alkaline earth metals such as calcium and magnesium, and metals such as aluminum). is included. Chondroitin sulfate is a glycosaminoglycan that exists as a sulfate ester with a disaccharide of D-glucuronic acid and N-acetyl-D-glucosamine as the main repeating unit. are also included in the chondroitin sulfate in the present invention. As chondroitin sulfate, those obtained from animals such as sharks, those obtained from microorganisms, commercially available products, and the like can also be used.

Royal jelly is a milky white jelly-like substance produced by mixing the secretions secreted from the hypopharyngeal and mammary glands of 3- to 12-day-old worker bees. Main physiologically active ingredients in royal jelly include, for example, organic acids such as 10-hydroxydecenoic acid (hereinafter referred to as "decenoic acid") unique to royal jelly, proteins, lipids, sugars, B vitamins, Vitamins such as folic acid, nicotinic acid and pantothenic acid, and various minerals are included. Royal jelly in the present invention includes fresh royal jelly, dried royal jelly, dried royal jelly powder, enzyme-treated royal jelly, royal jelly extract, fermented royal jelly and the like. Also, royal jelly may be produced in European countries, Oceania countries, America, Brazil, Japan, China, and other Asian countries.

Dried royal jelly powder is obtained by drying raw royal jelly and pulverizing it.

Enzyme-treated royal jelly is obtained by treating royal jelly with a protease (protease). Preferred is a hypoallergenic enzyme-treated royal jelly obtained by suppressing allergic reactions caused by proteins contained in royal jelly by protease treatment. Therefore, the enzyme-treated royal jelly may contain the above-mentioned organic acids such as decenoic acid, lipids, sugars, vitamins, and various minerals in addition to protease-degraded proteins contained in the royal jelly.

The royal jelly used for the production of the enzyme-treated royal jelly is not limited, but includes raw royal jelly, royal jelly powder obtained by drying and pulverizing raw royal jelly, and raw royal jelly extracted with water or hydrous ethanol. Enzyme-treated royal jelly can be produced according to the descriptions in JP-A-2007-295919, JP-A-2007-295920, and the like.

A royal jelly extract is obtained by extracting royal jelly (including raw, dried and pulverized products) with water, water-containing ethanol, or the like.

A fermented product of royal jelly can be produced by a conventional method using microorganisms such as yeast and lactic acid bacteria.

Although the above-mentioned royal jelly, glucosamine and chondroitin sulfate can be used as they are in the food composition of the present invention, minerals, vitamins, flavonoids, quinones, polyphenols, amino acids, nucleic acids, essential Fatty acids, cooling agents, binders, sweeteners, disintegrants, lubricants, coloring agents, fragrances, stabilizers, preservatives, sustained release modifiers, surfactants, solubilizers, wetting agents, etc. can be added. can.

The food composition of the present invention includes any food composition that can be ingested by animals (including humans). The type of the food composition of the present invention is not particularly limited, for example, dairy products; fermented foods (yogurt, royal jelly, etc.); Beverages containing lactic acid bacteria, yoghurt drinks, carbonated drinks, Japanese sake, western liquors, liquors such as fruit wines, etc.); Spreads (custard cream, etc.); Pastes (fruit paste, etc.)
Western sweets (chocolate, donuts, pies, cream puffs, gum, jelly, candy, cookies, cakes, puddings, etc.); candy, sherbet, etc.); foods (curry, beef bowl, porridge, miso soup, soup, meat sauce, pasta, pickles, jam, etc.); seasonings (dressing, sprinkle, umami seasoning, soup stock, etc.) be done.

The method for producing the food composition of the present invention is also not particularly limited, and any known method can be used as appropriate.

The food composition of the present invention can also be used as a health food, functional food, dietary supplement, supplement, health food, or specified health food. The dosage unit form when used as a supplement is not particularly limited and can be appropriately selected, and examples thereof include tablets, capsules, granules, liquids, powders and the like.

The total content of royal jelly, glucosamine and chondroitin sulfate in the food composition of the present invention is usually 30 to 85% by weight, preferably 50 to 85% by weight or 30 to 60% by weight. It is possible to

The intake amount of the food composition of the present invention can be appropriately set according to various conditions such as body weight, age, sex and symptoms of the intake person.

The food composition of the present invention is useful for prevention and/or treatment of bone weight loss diseases.

The agent for promoting differentiation into osteoblasts and the agent for promoting osteogenesis of the present invention may contain additional components as long as the effect of promoting differentiation into osteoblasts is not hindered. The total proportion of royal jelly, glucosamine and chondroitin sulfate in the osteoblast differentiation promoter and osteogenesis promoter when such additional components are included is, for example, 30 to 85% by weight.

The agent for promoting differentiation into osteoblasts and the agent for promoting osteogenesis of the present invention are not particularly limited in form, and examples thereof include powder, granule, tablet, capsule, liquid, suspension, emulsion, and the like. may have a formulation form of

The agent for promoting differentiation into osteoblasts and the agent for promoting osteogenesis of the present invention have an effect of promoting differentiation into osteoblasts, and therefore are suitable as active ingredients of pharmaceutical compositions for the treatment and/or prevention of bone weight loss diseases. can be used for

The agent for promoting differentiation into osteoblasts and the agent for promoting osteogenesis of the present invention are used in foods and drinks intended for health, maintenance, promotion, etc., such as health foods, functional foods, dietary supplements, supplements, and foods for specified health uses. , or the meaning of food with nutrient function claims. In addition, the osteoblast differentiation promoting agent and osteogenesis promoting agent of the present invention also include the meaning of an additive imparting an osteoblast differentiation promoting effect and an osteogenesis promoting agent effect.

The above food and drink may optionally contain excipients, brightening agents, minerals, vitamins, flavonoids, quinones, polyphenols, amino acids, nucleic acids, essential fatty acids, cooling agents, binders, sweeteners, Disintegrants, lubricants, colorants, fragrances, stabilizers, preservatives, sustained release modifiers, surfactants, solubilizers, wetting agents, etc. can be added.

The dosage unit form when used as a supplement is not particularly limited and can be appropriately selected, and examples thereof include tablets, capsules, granules, liquids, powders and the like.

The total proportion of royal jelly, glucosamine and chondroitin sulfate contained in the above food and drink can be, for example, 30 to 85% by weight.

The amount of intake of the above-mentioned food and drink can be appropriately set according to various conditions such as the body weight, age, sex and symptoms of the intake person.

When preparing as a pharmaceutical composition, the royal jelly, glucosamine and chondroitin sulfate are used as they are, or tablets (uncoated tablets, sugar-coated tablets, effervescent tablets, film-coated tablets, chewable tablets, lozenges, etc.), capsules, pills, powders (powders), fine granules, granules, liquids, suspensions, emulsions, syrups, pastes, injections ( At the time of use, it can be prepared into a form such as distilled water or an infusion solution such as an amino acid infusion solution or an electrolyte infusion solution to prepare a liquid preparation) to prepare a pharmaceutical preparation.

The total content of royal jelly, glucosamine and chondroitin sulfate in the pharmaceutical composition of the present invention is usually 30 to 85% by weight, preferably 50 to 85% by weight or 30 to 60% by weight. It is possible to

The dosage of the pharmaceutical composition of the present invention can be appropriately determined according to various conditions such as patient body weight, age, sex and symptoms.

The pharmaceutical composition of the present invention is useful for prevention and/or treatment of bone weight loss diseases.

Bone weight-lowering diseases include, but are not limited to, osteoporosis (primary osteoporosis and secondary osteoporosis), cancer bone metastasis, hypercalcemia, Paget's disease, bone defect, osteonecrosis, and the like. is mentioned. In addition, the food composition, differentiation promoter, osteogenesis promoter and pharmaceutical composition of the present invention are thought to be applicable to promotion of bone formation after bone surgery, bone graft replacement therapy, and the like.

The food composition, differentiation promoter, osteogenesis promoter and pharmaceutical composition of the present invention preferably contains 1 to 20 parts by weight of glucosamine and 0.01 part of chondroitin sulfate per 1 part by weight (dry weight) of royal jelly. ~10 parts by weight, more preferably 4-20 parts by weight of glucosamine and 0.2-10 parts by weight of chondroitin sulfate, or 1-12 parts by weight of glucosamine and 0.01-2 parts by weight of chondroitin sulfate.

The food composition, differentiation promoter, osteogenesis promoter, and pharmaceutical composition of the present invention described above are applicable to mammals including humans.

Test examples are given below to describe the present invention in more detail. However, the present invention is by no means limited to these test examples.

[Example 1]
A mixture of royal jelly, glucosamine and chondroitin sulfate:
Phenol red-free 10% FBS-added α-MEM medium (Invitrogen, Cat. No. 41061-029) and filter-sterilized with a Millex-HV Filter Unit PVDF 0.45 μm (Millipore, SLHV033RS).

[Comparative Example 1]
The 10% FBS-added α-MEM medium containing no test substance was prepared.

[Comparative Example 2]
Compositions containing only royal jelly as test substance:
The freeze-dried powder of royal jelly was dissolved in the 10% FBS-added α-MEM medium to a final concentration of 100 µg/mL, and sterilized with a filter.

[Comparative Example 3]
A mixture of glucosamine and chondroitin sulfate:
Glucosamine and chondroitin sulfate were dissolved in the above 10% FBSα-MEM medium to final concentrations of 1149 µg/mL and 135 µg/mL, respectively, and filter sterilized.

[Test Example 1]
(1) Test method Osteoblast MC3T3-E1 (Riken Cell Bank, RCB1126) was suspended in 10% FBS-added α-MEM medium to a concentration of 2.5 × 10 ⁵ cells/mL, and 500 μl was added to a 48-well plate. sown. The next day, the medium was replaced with 200 μl of the medium of Example 1 and Comparative Examples 1-3. The cells were cultured for 21 days, during which the media of Example 1 and Comparative Examples 1-3 were replaced at intervals of 3-4 days. After culturing for 21 days, osteoblast differentiation was evaluated by measuring the amount of osteocalcin, an osteoblast differentiation marker, by ELISA.

Osteocalcin is a protein produced by osteoblasts, and its expression level is known to increase when osteoblasts mature. Non-Patent Document 1 also evaluates the same protein as a marker indicating osteoblast differentiation, and the amount of osteocalcin is generally used as an index indicating osteoblast maturation.

(2) Test Results FIG. 1 shows the amount of osteocalcin after each test sample was added to MC3T3-E1 and cultured for 21 days. As can be seen from this, the addition of royal jelly, glucosamine, and chondroitin sulfate significantly increased the amount of osteocalcin compared to the case where nothing was added. Furthermore, the addition of royal jelly, glucosamine and chondroitin sulfate significantly increased the amount of osteocalcin compared to the addition of royal jelly alone or glucosamine and chondroitin sulfate alone. From the above results, it was found that the simultaneous addition of royal jelly, glucosamine and chondroitin sulfate synergistically increased the amount of osteocalcin compared to the case of adding only royal jelly or only glucosamine and chondroitin sulfate.

[Test Example 2]
(1) Test method Osteoclast RAW264.7 (Riken Cell Bank, RCB0535) was suspended in 10% FBS-added α-MEM medium (Invitrogen, Cat. No. 41061-029) at 1 × 10 ⁴ cells/ml. , 200 μl were dispensed into a 96-well plate. The next day, the medium was replaced with 200 µl of the medium of Example 1 and Comparative Example 1 to which RANK Ligand (RANKL) was added to a final concentration of 50 ng/ml. After culturing for 3 days, the medium was replaced with 200 μl of the medium of Example 1 and Comparative Example 1 containing RANKL, and cultured for an additional 2 days. After washing the culture plate with PBS, fix the cells with 10% formalin solution, perform TRAP staining (tartaric acid-resistant acid phosphatase staining), and count the number of TRAP-positive osteoclasts with 3 or more nuclei stained red. osteoclast differentiation was assessed.

TRAP is a protein produced by osteoclasts, and is known to become TRAP-positive and multinucleate when osteoclasts differentiate. Also in Non-Patent Document 2, the number of cells expressing the same protein is counted as a marker indicating osteoclast differentiation, and the number of TRAP-positive cells is generally used as an index indicating osteoclast maturation.

(2) Test Results FIG. 2 shows the number of TRAP-positive cells with 3 or more nuclei obtained by adding each test sample to RAW264.7, culturing it, and then TRAP-staining it. As can be seen from this, it was found that the addition of royal jelly, glucosamine and chondroitin sulfate significantly suppressed the increase in the number of TRAP-positive cells compared to the case where nothing was added.

[Prescription example]
Formulation examples of the food composition of the present invention are shown below.

Formulation example 1
Dry royal jelly powder: 25% by weight, shark cartilage extract (containing chondroitin sulfate): 6% by weight, glucosamine hydrochloride: 58% by weight, MSM, cellulose, sucrose fatty acid ester, fine silicon dioxide, hyaluronic acid, chicken cartilage extract , Hihatsu extract powder and thickener combined 10% by weight
are mixed and compressed to obtain tablets, which are then coated with a mixed solution containing appropriate amounts of zein: 1% by weight, glycerin, water and alcohol.

Formulation example 2
Glucosamine hydrochloride: 61% by weight, shark cartilage extract (containing chondroitin sulfate): 7% by weight,
Dry royal jelly powder: 5% by weight, 16% by weight of hyaluronic acid, chicken cartilage extract, cellulose, fine silicon dioxide, sucrose fatty acid ester, thickener and vitamin D3 were mixed and compressed into tablets. After that, it is coated with 11% by weight of a mixture of yeast cell walls, coral calcium, glycerin, water, alcohol and a thickener.

Claims (2)

A food composition containing royal jelly, glucosamine and chondroitin sulfate, wherein 1 part by weight of royal jelly contains 4 to 20 parts by weight of glucosamine and 0.2 to 10 parts by weight of chondroitin sulfate, and the sum of royal jelly, glucosamine and chondroitin sulfate is 30 to 85% by weight of the total amount of the food composition (excluding beverages and food compositions for promoting adiponectin secretion). A food composition containing royal jelly, glucosamine and chondroitin sulfate, wherein 1 part by weight of royal jelly contains 4 to 20 parts by weight of glucosamine and 0.2 to 10 parts by weight of chondroitin sulfate, and the sum of royal jelly, glucosamine and chondroitin sulfate is 30 to 85% by weight of the total amount of the food composition (excluding beverages, and the composition contains corosolic acid, resveratrol, isoflavone, artepillin C, 3,4-dihydroxy- 5-prenylcinnamic acid, quercetin, kaempferide, p-coumaric acid, 4-dihydrocinnamoyloxy-3-prenylcinnamic acid, propolis, astaxanthin, lycopene, anthocyanin, proanthocyanidin, catechins, isorhamnetin, kaempferol, rutin, coenzyme Q10, vitamin E, and one or more antioxidants selected from calcium ascorbate).

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