JP4659405B2 - Bone formation promoter - Google Patents

Description

  The present invention relates to an osteogenesis promoter containing a fourth component of complement and / or β-defensin as an active ingredient. In addition, the present invention relates to a bone formation promoting food / beverage product, medicine or feed containing a complement fourth component and / or β-defensin.

  In recent years, with the aging of the population, various bone diseases such as osteoporosis, fractures, and back pain tend to increase. In bone tissue, bone formation and bone resorption are constantly carried out, and the balance between bone formation and bone resorption is maintained when young, but the balance tends to bone resorption due to various causes (aging) Coupling). If this state continues for a long period of time, the bone tissue becomes brittle, and various bone diseases such as osteoporosis, fractures, and back pain occur. If this uncoupling can be prevented, it is considered that various bone diseases such as osteoporosis, fracture, and back pain can be prevented.

Conventionally, as a method for preventing uncoupling and preventing or treating various bone diseases, (1) calcium supplementation with diet, (2) light exercise, (3) sunbathing, (4) drug treatment, and the like have been performed. . For calcium supplementation with diet, calcium salts such as calcium carbonate and calcium phosphate, and natural calcium preparations such as eggshell and fish bone powder are used. However, these are not necessarily materials suitable for oral intake. Light exercise is good for jogging or taking a walk, but if the body is weak, light exercise is troublesome, and even a bedridden elderly person can hardly exercise. Sunbathing is considered good in terms of supplementing activated vitamin D3, but this alone is not sufficient. For drug administration, 1α-hydroxyvitamin D 3, calcitonin preparations and the like are used, and it is known that they are effective for the treatment of osteoporosis. However, these substances are medicines themselves and cannot be used as food materials.

  In order to obtain a substance having a bone formation promoting action and a bone resorption inhibiting action that can be used as a food material, the present inventors have continued to search for a bone formation promoting factor and a bone resorption inhibiting factor present in milk. In the process, the present inventors have found that the complement fourth component and β-defensin have an osteogenesis promoting action by promoting osteoblast differentiation. Osteoblasts are cells that play a central role in bone formation, are present on the surface of bone tissue, and secrete bone matrix proteins. Calcium phosphate crystals are deposited on the bone matrix protein, resulting in a hard bone tissue.

  On the other hand, osteoclasts are cells that originate from hematopoietic stem cells, exist on the surface of cancellous bone, and dissolve bone. It is believed that osteoclasts dissolve bone matrix (bone resorption), and then osteoblasts synthesize bone matrix, resulting in bone formation and growth (modeling) and metabolism (remodeling). The present invention finds that the fourth component of complement and / or β-defensin has an osteogenesis promoting action by promoting the differentiation of osteoblasts that play a central role in bone formation in this bone metabolism. It is a thing.

  Complement component C4 is an enzyme-like substance that exists in serum and is activated by reacting with an antigen-antibody complex and exerts physiological activity while causing a complex reaction. The complement fourth component itself has no specificity for the antigen, but is activated by the antigen-antibody complex to cause an increase in phagocytosis and destroy cells and bacteria to which the specific antibody is bound. Currently, nine components are known from the first complement component (C1) to the ninth complement component (C9). The fourth complement component is a protein having a molecular weight of 210 kDa consisting of three types of polypeptide chains: α chain, β chain, and γ chain. Embryos / fetuses containing the third component of complement as an active ingredient, and their nutrition factors composition for tissue culture and compositions for treating infertility have been disclosed (see, for example, Patent Document 1). The four components were not known to promote osteoblast differentiation.

Defensin is known as a strong basic antibacterial peptide having three S—S bonds in the molecule, and consists of 29 to 34 amino acids with 4 to 10 arginine residues and 6 cysteine residues. It is a basic protein. Due to the difference in the position of cysteine residues, it can be divided into α and β types. In addition to antibacterial activity against gram-positive and gram-negative bacteria, it possesses antibacterial activity against molds and viruses. β-defensins are expressed in respiratory epithelial cells and mucosal epithelial cells, and six types are known in humans. In addition to antibacterial activity, defensin is known to be widely involved in biological defense such as cytotoxicity against normal cells and cancer cells, histamine release from mast cells, and monocyte chemotaxis. From a retroviral infection therapeutic agent (for example, see Patent Document 2) consisting of administering a specific culture filtrate of yeast and lactic acid bacteria to secrete defensin and the like, and from mugwort roots that stimulate the expression of defensin in the skin Such a cosmetic composition has been disclosed (for example, see Patent Document 3), but it has not been known for promoting differentiation of osteoblasts.
Japanese Patent Laid-Open No. 10-033164 JP 2004-115497 A JP 2004-067660 JP

  Due to the nature of various bone diseases such as osteoporosis, fractures, and back pain, it can be taken orally in the long-term and directly in the daily diet, with no problems in taste, and directly promotes bone formation. Providing a new bone formation promoter and a bone formation promoting food and beverage, medicine or feed that can be expected to prevent or improve various bone diseases such as osteoporosis, fractures, and back pain. Let it be an issue.

In the process of searching for a substance having a bone formation promoting action and a bone resorption preventing action, the present inventors promoted osteoblast differentiation by promoting the differentiation of osteoblasts by the fourth component of complement and / or β-defensin. It has been found that it has an accelerating action, and the present invention has been completed.
Bone consists of bone minerals such as calcium and minerals and bone matrix such as collagen. In order to strengthen the bone, not only the amount of bone mineral evaluated by an index such as bone density but also the formation of a bone matrix that becomes the basis of a supple and elastic bone is important. Since collagen is produced by differentiated osteoblasts, the promotion of collagen production by osteoblasts leads to the formation of bone matrix, which can lead to an increase in bone strength.
Therefore, the present invention is to provide a novel osteogenesis promoter containing the fourth component of complement and / or β-defensin as an active ingredient. Moreover, this invention is providing the food / beverage products, the pharmaceutical, or the feed which mix | blended the complement 4th component and / or (beta) defensin, and provided the bone formation promotion effect | action.
Complement fourth component and / or β-defensin can be prepared from blood or milk. The fourth component of complement is commercially available and is sold by Sigma, etc. Β-defensins are also commercially available and are sold by Peptide Institute, Inc.

  The osteogenesis promoter containing the fourth component of complement and / or β-defensin of the present invention as an active ingredient, and the bone formation-promoting food, beverage, medicine, feed, etc. containing the complement fourth component and / or β-defensin, By promoting the differentiation of osteoblasts, it has an effect of promoting osteogenesis and is useful for the prevention and improvement of various bone diseases such as osteoporosis, fractures, and back pain. In addition, the osteogenesis promoter and osteogenesis promoting food / drink, medicine, feed, etc. of the present invention are easy to obtain raw materials, always have a certain quality, and are inexpensively manufactured without requiring complicated processes. And can be prepared in large quantities.

  When administering the osteogenesis promoter of the present invention, the complement fourth component and / or β-defensin is shaped with lactose, starch or the like according to a conventional method, if necessary, and then powder, granule, tablet It can also be used as a capsule or formulated as a drink. Furthermore, this complement fourth component and / or β-defensin is blended in foods and drinks such as milk, milk drink, coffee drink, juice, jelly, biscuits, bread, noodles, sausage, etc. to differentiate osteoblasts. By promoting, it is possible to promote bone formation. Furthermore, the bone formation promoting action can be further enhanced by using in combination with a calcium agent having good absorbability such as calcium chloride, calcium carbonate, calcium lactate, eggshell, and milk-derived calcium. Moreover, these active ingredients can be contained in feed to promote bone formation in livestock and poultry.

In order to obtain an osteogenesis promoting effect, as an effective amount of the osteogenesis promoter and osteogenesis promoting food and drink of the present invention, the fourth component of complement and / or β-defensin 1 ng to 100 mg is used several times a day in adults. Orally divided into two.
Thus, by ingesting the osteogenesis promoter, osteogenesis promoting food and drink of the present invention, and medicine, osteoblast differentiation is promoted to promote osteogenesis and prevent or prevent various bone diseases such as osteoporosis. Can be improved. The fourth complement component and β-defensin were originally blood or milk-derived components, and no acute toxicity was observed in rats.
EXAMPLES Examples and test examples are shown below, and the present invention will be described in more detail. However, these are merely illustrative, and the present invention is not limited by these.

Test example 1

The effects of complement fourth component and β-defensin on osteoblast differentiation were examined. Specifically, human-derived preosteoblast MG63 cells were seeded in a 96-well plate at 2 × 10 ⁴ / ml using DMEM medium (Flow Laboratories) containing 10% fetal bovine serum, and 5% The cells were cultured at 37 ° C. for 4 days in the presence of CO _{2 to} obtain cultured cells for testing. Then, the medium is replaced with a medium containing 1% fetal bovine serum, and the complement fourth component (Complement C4, C8195, manufactured by Sigma) is adjusted to a final concentration of 0.1, 1, and 10 μg / ml, and β Defensins (β-defensins 1 and 2, manufactured by Peptide Institute, Inc.) were added to the medium so as to have final concentrations of 0.1, 1, and 10 μg / ml, and cultured at 37 ° C. for 5 days. The culture supernatant was collected, and osteoblast differentiation promoting activity was examined by measuring the amount of type I collagen in the culture supernatant using Procollagen Type I C-peptide EIA Kit (Takara MK101, Takara Shuzo). As a control, a supplemental fourth component and β-defensin-free additive were used. The amount of collagen production was expressed as the ratio (%) of the measured amount of type I collagen in each sample to the measured amount of collagen type I. The results are shown in Table 1.

[Table 1]
-------------------------------------------------- ------------------
Final concentration (μg / ml) Collagen production (%)
-------------------------------------------------- ------------------
Control (no additive)-100 ± 6
Complement 4th component 0.1 138 ± 7
1 169 ± 15
10 161 ± 9
β-defensin 1 0.1 127 ± 11
1 183 ± 9
10 197 ± 18
β-defensin 2 0.1 134 ± 9
1 147 ± 5
10 177 ± 14
-------------------------------------------------- ------------------

  All the groups supplemented with complement fourth component and β-defensin have increased type I collagen content compared to the control (no complement fourth component and β-defensin added) group, and have osteoblast differentiation promoting action I understood.

(Production of complement fourth component and β-defensin)
After passing 10 l of defatted milk through a S-Sepharose column manufactured by Pharmacia, it was thoroughly washed with 20 mM phosphate buffer (pH 7). Gradiently elutes the adsorbed protein while increasing the ratio of phosphate buffer (pH 8.5) containing 1.5M salt to 100% in 1 hour. 0.3 mg of β-defensin was recovered at 20 minutes.
The complement fourth component and β-defensin thus obtained can be used as is as an osteogenesis promoter.

(Manufacture of osteogenesis promoter)
Complement C4 (Complement C4, C8195, manufactured by Sigma) 1mg, water-containing crystalline glucose 93.4g, calcium carbonate 5g, sugar ester 1g, fragrance 0.5g added, mixed, and then tableted into tablets. The osteogenesis promoter of the present invention was produced.

(Manufacture of osteogenesis promoter)
Add 13.4 mg of β-defensin (β-Defensin-1,4337-s, manufactured by Peptide Laboratories) to 93.4 g of hydrous crystalline glucose, 5 g of calcium carbonate, 1 g of sugar ester, and 0.5 g of fragrance, and mix them into a tablet. Tableting was performed to produce the osteogenesis promoter of the present invention.

(Manufacture of osteogenesis promoter)
Complement fourth component (Complement C4, C8195, manufactured by Sigma) 1 mg, β-defensin (β-Defensin-2, 4338-s, manufactured by Peptide Institute, Inc.) 1 mg, hydrous crystalline glucose 93.4 g, calcium carbonate 5 g Then, 1 g of sugar ester and 0.5 g of fragrance were added and mixed, and then tableted to produce the osteogenesis promoter of the present invention.

(Manufacture of milk beverages for promoting bone formation)
After homogenizing raw milk at a homogeneous pressure of 120 kg / cm ² , pasteurize the fourth component of complement (Complement C4, C8195, Sigma) to 10 mg per liter in raw milk that has been heat-sterilized at 75 ° C for 15 seconds. It was added under aseptic condition and filled into a 100 ml capacity glass bottle to produce a milk beverage for promoting bone formation.

(Manufacture of milk beverages for promoting bone formation)
After homogenizing raw milk at a homogeneous pressure of 120 kg / cm ² , beta defensin (β-Defensin-1,4337-s, manufactured by Peptide Institute, Inc.) It was added aseptically to 10 mg, and filled into a 100 ml capacity glass bottle to produce a milk beverage for promoting bone formation.

(Manufacture of bone formation promoting jelly)
Fructose 20.0 (wt%), Granulated sugar 15.0 (wt%), Minamata 5.0 (wt%), Agar 1.0 (wt%), Fragrance 0.11 (wt%), Calcium 0.1 (wt%), Water 58.39 (wt%) After mixing the raw materials at a ratio and sterilizing by heating, the fourth component of complement (Complement C4, C8195, manufactured by Sigma) is added under aseptic conditions to 10 mg per liter and filled into a container. A accelerating jelly was produced.

(Manufacture of infant formula for bone formation promotion)
Nonfat milk 75.61 (wt%), whey protein concentrate 2.36 (wt%), lactose 13.86 (wt%), mineral mixture 0.32 (wt%), water-soluble vitamin mixture 0.32 (wt%), fat containing fat-soluble vitamins The raw materials were mixed and sterilized at a ratio of 7.53 (% by weight), concentrated, and spray-dried to produce a raw material powdered milk powder. Add β-defensin (β-Defensin-2, 4338-s, manufactured by Peptide Laboratories, Inc.) under aseptic conditions to 10 mg / kg, mix, and prepare infant formula for bone formation promotion. Manufactured.

(Manufacture of dog food for promoting bone formation)
Soybean cake 12.0 (wt%), skim milk powder 14.0 (wt%), soybean oil 4.0 (wt%), corn oil 2.0 (wt%), palm oil 28.0 (wt%), corn starch 15.0 (wt%), wheat flour 9.0 (Wt%), bran 2.0 (wt%), vitamin mixture 9.0 (wt%), mineral mixture 2.0 (wt%), cellulose 3.0 (wt%) The fourth component (Complement C4, C8195, manufactured by Sigma) was added under aseptic conditions so as to be 10 mg per liter to form a dog breeding feed for promoting bone formation (dog food).

Claims (2)

A bone formation promoter comprising a fourth component of complement and / or β-defensin. A bone formation-promoting pharmaceutical comprising a fourth component of complement and / or β-defensin and a component acceptable for formulation .