JP5379884B2 - Bone resorption inhibitor - Google Patents

Description

The present invention includes β ₂ microglobulin, histone, complement third component, monocyte chemotactic protein 1
, A lysozyme and a ribonuclease. The present invention also provides a bone resorption-suppressing food / drink, pharmaceutical or feed containing at least one of β ₂ microglobulin, histone, complement third component, monocyte chemotactic protein 1, lysozyme, and ribonuclease. About.

In recent years, various bone diseases such as osteoporosis, fractures, and back pain tend to increase as the elderly population increases. 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 methods for preventing uncoupling and preventing or treating various bone diseases, (1) dietary calcium supplementation, (2) light exercise, (3) sunbathing, (4) drug treatment, and the like have been performed. Yes. 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 active vitamin D ₃ , but it is not enough. For drug administration, 1α-hydroxyvitamin D ₃ , calcitonin preparation 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.

On the other hand, the present inventors have continued to search for bone strengthening and bone resorption inhibiting factors present in whey protein in order to obtain a substance having bone strengthening action and bone resorption inhibiting action that can be used as a food material. . As a result, it was found that a protein and peptide mixture obtained by removing whey-derived salts from the water-soluble fraction of whey protein by treatment such as reverse osmosis membrane or electrodialysis has a bone strengthening action (for example, Patent Document 1). reference.). And, it was found that the fraction obtained by subjecting the aqueous solution of the protein and peptide mixture to ethanol treatment, heat treatment, salt treatment, and ultrafiltration membrane treatment has an osteoblast proliferation promoting action and a bone strengthening action (for example, (See Patent Documents 2 and 3.)
Moreover, it discovered that the basic protein which exists only in trace amount in milk has an osteoblast proliferation promoting effect, a bone strengthening effect, and a bone resorption preventing effect (for example, refer patent document 4).

The present inventors have further pursued, tried to separate and purify a component having a bone resorption inhibitory action, and identified the substance. As a result, known substances β ₂ microglobulin, histone, complement third component, monocyte It was confirmed that it was chemotaxis protein 1, lysozyme, and ribonuclease.
And it discovered that these substances could be utilized as a bone resorption inhibitor, and came to complete this invention.

β ₂ microglobulin is a protein with a molecular weight of about 11.6 kDa and is a major histocompatibility complex (
MHC). It is known to be widely distributed in whole body tissues including blood, and it has been reported that it is also present in milk in humans and cows.

Regarding the action of β ₂ microglobulin on bone, as a marker of bone turnover, it has been reported that high rotation type osteoporosis has a high blood β ₂ microglobulin concentration (for example,
See Non-Patent Document 1. ). In addition, β ₂ microglobulin has been reported to promote bone formation because it promoted the growth of cultured osteoblast MC3T3 (see, for example, Non-Patent Document 2). on the other hand,
Regarding bone resorption, there is a report that β ₂ microglobulin promotes calcium elution from bone (see, for example, Non-Patent Document 3). However, the action of promoting calcium elution is different from the action of suppressing bone resorption, and there is no literature describing the action of β ₂ microglobulin that suppresses bone resorption as found by the present inventors.

Histone is a basic protein present in the cell nucleus of higher animals. In higher animals, DNA (deoxyribonucleic acid) that controls genetic information is said to have a length of about 2 m in a linear distance, but it is an essential protein for efficiently storing the DNA in the cell nucleus.
That is, DNA is functionally folded while wrapped around histones. Usually, H1, H2A
, H2B, H3, and H4, and each H2A, H2B, H3, and H4 molecules each form a single aggregate of 8 molecules. DNA is wrapped around the assembly to form a unit called chromatin. H
1 is responsible for bonding chromatin together. In this way, histone and DNA are always a set, and are present in the nucleus at a ratio of approximately 1: 1 by weight.

There have been many reports on the nutritional effects of nucleic acids and their constituents, and there is a view that nucleic acids are the sixth essential nutrient. However, there is no report of nutritional effects on histones that exist in pairs. Animal food is basically a collection of cells. So, as long as you eat, histones are a protein you will inevitably consume regardless of the type of animal. However, in the case of human beings, cell-free foods using food materials that are refined and do not contain cell nuclei, such as white rice, flour, and sugar, are very often consumed. If only histone promotes bone formation or suppresses bone resorption, it is sufficient to incorporate a large amount of food ingredients such as liver and white larvae, which are cell masses, but there are significant taste and food processing problems. The restrictions are extremely large.

Complement is an enzyme-like substance that exists in serum, is activated by reacting with an antigen-antibody complex, and exhibits physiological activity while causing a complex reaction. Currently, nine components from C1 to C9 are known. Complement component 3 corresponds to C3 and is called a complement third component, and has the largest content among complement components. Its structure is a glycoprotein in which an α chain with a molecular weight of 105,000 and a β chain with a molecular weight of 75,000 are cross-linked by two SS bonds. This C3 with a molecular weight of 180,000 is divided into C3a with a molecular weight of 9,000, which is obtained by cleaving a part of the α chain by C3 convertase, and the remaining C3b with a molecular weight of 170,000 including two SS bonds. C3a is known as one of the typical anaphylatoxins that cause an inflammatory reaction, such as stimulating mast cells to promote histamine release. C3b is also C
3 Degraded by convertase, part of which is covalently bound to the antigen-antibody complex. Since phagocytic cells such as macrophages in the blood have a C3b receptor on the cell surface, this reaction eventually enhances phagocytosis of foreign substances by phagocytic cells.

In this way, the third component of complement (C3) is a substance that is deeply involved in the immune response. It has been clarified by the present invention that it plays a suppressing role.

Monocyte chemotactic protein-1 (Monocyte chemotactic protein-1 or Monocyte Chemoattr
actant protein-1, MCP-1) is a monocyte chemotactic factor produced by monocytes, vascular endothelium, glioma cell lines and the like. RANTES (regulated on activation, normal T cell expressed and secreted
) And a representative CC chemokine family with a molecular weight of 8,000 to 18,000 and 76 amino acids. It is mainly chemotactic for monocytes and does not act on neutrophils or lymphocytes.

Lysozyme is an enzyme that hydrolyzes the β-1 → 4 bond between N-acetylmuramic acid and N-acetylglucosamine present in mucopeptides of bacterial cell walls and is widely distributed in the animal and plant kingdoms. . There are four groups based on substrate specificity and molecular weight.

Ribonuclease is a general term for enzymes that act on ribonucleic acid (RNA), which is a kind of nucleic acid, to produce mononucleotides or oligonucleotides.

As described above, β ₂ microglobulin has been reported to promote bone formation, but histones other than β ₂ microglobulin, complement third component, monocyte chemotactic protein 1, lysozyme, and ribonuclease, In any case, there is no literature describing the action of suppressing bone resorption as well as literature promoting bone formation.

Japanese Unexamined Patent Publication No. 4-183371 Japanese Patent Laid-Open No. 5-177715 Japanese Patent Laid-Open No. 5-320066 JP-A-8-15131

JM Quesada and six others, “Serum β2 microglobulin as a marker for high bone remodeling in older women”, Mechanisms of Aging and Development, (USA) 1998, 102 No., p.293-298 E. Balint and two others, “The role of interleukin 6 in mineral elution from bone induced by β2 microglobulin”, Kidney International, (USA), 2000, 57, p. 1599- 1607 J. Petersen et al., “In vivo effect of β2 microglobulin on bone resorption”, American Journal of Kidney Diseases, (USA), 1994, 23, p. 726- 730

An object of the present invention is to obtain a bone resorption inhibitor which can be used as a food material and can be taken orally for a long period of time and is a safe substance and has a bone resorption inhibitory action. Moreover, this invention makes it a subject to obtain the food-drinks, pharmaceuticals, or feed for bone resorption suppression.

In view of these problems, the present inventors have eagerly and continuously searched for substances that show bone strengthening action widely contained in food materials, and promote osteoblast proliferation promotion action, bone strengthening action, and bone resorption prevention action. We tried to isolate and purify a component having a bone growth promoting action and a bone resorption-preventing action, and identified the substance. Β ₂ microglobulin, histone, complement third component, monocyte running It was confirmed that the protein was categorized protein 1, lysozyme, and ribonuclease. And it discovered that these substances can be utilized as a bone resorption inhibitor, and came to complete this invention.

That is, the present invention relates to a bone resorption inhibitor comprising one or more of β ₂ microglobulin, histone, complement third component, monocyte chemotactic protein 1, lysozyme, and ribonuclease as an active ingredient.

The present invention also provides a bone resorption-suppressing food / drink, pharmaceutical or feed containing at least one of β ₂ microglobulin, histone, complement third component, monocyte chemotactic protein 1, lysozyme, and ribonuclease. About.

Β ₂ microglobulin of the present invention, histone, complement third component, monocyte chemotactic protein 1,
A bone resorption inhibitor comprising one or more of lysozyme and ribonuclease as an active ingredient is useful for the prevention and improvement of various bone diseases such as osteoporosis by oral administration thereof.
Moreover, when these active ingredients are blended in foods, drinks, medicines, feeds, etc., there is an effect of suppressing and improving various bone diseases such as osteoporosis by suppressing bone resorption.

Osteoclasts are cells that originate from hematopoietic stem cells and are present on the surface of cancellous bone and lyse 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 has been found that bone resorption by osteoclasts is remarkably suppressed by β ₂ microglobulin, histone, complement third component, monocyte chemotactic protein 1, lysozyme, and ribonuclease. .

Inclining bone metabolism to a balance of bone formation by using the bone resorption inhibitory action found in β ₂ microglobulin, histone, complement third component, monocyte chemotactic protein 1, lysozyme, and ribonuclease Can be expected. Therefore, the present invention provides a bone resorption inhibitor having a bone resorption inhibitory action, food and drink, medicine or feed. In the present invention, β ₂ microglobulin, histone, complement third component, simple substance Globe chemotaxis protein 1, lysozyme, and ribonuclease are all present in trace amounts in milk such as cows, buffalos, humans, pigs, sheep, goats and horses, and can be separated and extracted from milk. Milk components include raw milk, skim milk, or protein fraction separated from whey,
Alternatively, a cell fraction in milk separated from raw milk can be used. β ₂ microglobulin, complement third component, and monocyte chemotactic protein 1 can also be prepared from healthy livestock blood. As a material for extracting histones, cereal germs, white eggs and the like can be used in addition to milk components. As the grain germ, wheat germ, rice germ and the like can be used. Shiroko is salmon, trout, cod, herring,
What is collected from seafood such as squid and scallops may be used, but in particular, it is preferable to use the white larvae of fish such as salmon, trout and cod in terms of effective use of resources. .

β ₂ microglobulin, histone, complement third component, monocyte chemotactic protein 1, lysozyme, and ribonuclease are all commercially available, and these commercially available products can also be used. Moreover, you may use the recombinant goods produced by genetic engineering.

In addition, β ₂ microglobulin, histone, complement third component, monocyte chemotactic protein 1,
Lysozyme and ribonuclease can be trypsin, pancreatin, chymotrypsin,
What decomposed | disassembled by proteolytic enzymes, such as pepsin, papain, kallikrein, cathepsin, thermolysin, V8 protease, may be used.

For preparation from milk, for example, fresh milk is contacted with an ion exchange resin to obtain a fraction containing β ₂ microglobulin, histone, complement third component, monocyte chemotactic protein 1, lysozyme, and ribonuclease. Adsorption and elution by gradually increasing the concentration of sodium chloride, followed by gel filtration chromatography with β ₂ microglobulin, histone, complement third component,
Monocyte chemotactic protein 1, lysozyme, and ribonuclease can be obtained, respectively.

For preparation from blood, citrated plasma is reacted with barium citrate to recover β ₂ microglobulin adsorbed on insoluble barium, complement third component, and monocyte chemotactic protein 1 precipitate, respectively. Furthermore, the purity can be increased by ion exchange chromatography.

The bone resorption inhibitor of the present invention contains at least one of β ₂ microglobulin, histone, complement third component, monocyte chemotactic protein 1, lysozyme, and ribonuclease as an active ingredient. In addition, any one or more of β ₂ microglobulin, histone, complement third component, monocyte chemotactic protein 1, lysozyme, and ribonuclease are mixed with milk, milk beverage, coffee beverage, juice, jelly, biscuit, It may be blended in foods and beverages such as bread, noodles and sausages, and may be used as pharmaceuticals such as tablets and powders. In addition, in combination with calcium agents with good absorbability such as calcium chloride, calcium carbonate, calcium lactate, eggshell, milk derived calcium,
By tilting bone metabolism to the balance of bone formation, the bone strengthening action can be further enhanced.

The dose of the bone resorption inhibitor of the present invention varies depending on the active ingredient, age, therapeutic effect and disease state, but in the case of an adult, it may be taken orally in an amount of 1 ng to 1 g per day. Thus, by taking the bone resorption inhibitor of the present invention, various bone diseases such as osteoporosis can be prevented. Β ₂ microglobulin, histone, complement third component, monocyte chemotactic protein 1, lysozyme, and ribonuclease and its enzymatic degradation products are originally components derived from plasma and milk, and acute toxicity in rats Was not recognized. Moreover, these active ingredients can be contained in feed to suppress bone resorption of livestock and poultry.

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.

Known methods (Hoshi F., D. Nagai, S. Higuchi, T. Noso, A. Takahashi, S. Kawamura,
Veterinary Immunology and Immunopathology, 53, 29-38, 1996. Purification of bov
β ₂ microglobulin was prepared from bovine colostrum according to ine beta2-microglobulin from colostrums and its complete amino acid sequence.

That is, skim milk was obtained from 10 l of bovine colostrum by centrifugation, the pH was adjusted to 4 with 1N hydrochloric acid, and aggregated casein was precipitated by centrifugation. Diethylaminoethyl (DEAE) in which 20 g of casein was dissolved in 1 l of 5 mM sodium phosphate buffer (PBS: pH 8.3) and equilibrated with the same buffer
It was made to adsorb | suck to a cellulose (DE-52: made by Whatman) column. After washing with equilibration buffer, the obtained fraction was analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and the fraction containing 12 kDa protein was concentrated by ultrafiltration, and 5 mM PBS ( Dialyzed overnight at 4 ° C against pH 8.3). The concentrate was passed through a gel filtration chromatography consisting of a Sephadex G-75 column equilibrated with 5 mM PBS (pH 8.3). Concentrate the fraction containing 12 kDa protein by ultrafiltration,
Dialyzed against 10 mM ammonium acetate solution. This was freeze-dried to obtain 12 mg of β ₂ microglobulin which is an active ingredient of the present invention. The beta ₂ -microglobulin 12 mg was mixed with lactose 120 mg, to obtain a bone resorption inhibiting agent molded to the present invention into granules.

Histone, complement third component, monocyte chemotaxis protein 1, lysozyme, and ribonuclease can also be prepared from bovine colostrum by the same method as for β ₂ microglobulin.

After washing 1 kg of salmon roe (containing 6% nucleic acid) from which the skin has been removed, it is dehydrated and then water 3
1 was added and a homogenate was prepared with a homogenizer. Add 0.05 N sodium hydroxide solution to this homogenate to adjust the pH to 6, add 20 g of papain, degrade protein while stirring at 35 ° C for 2 hours, and then hold at 80 ° C for 30 minutes to lose the enzyme. The decomposition was terminated by activating.
After completion of the digestion, the precipitate was removed by centrifugation, and 2.5 times the amount of 95% ethanol was added to the resulting supernatant to precipitate the DNA. The aqueous ethanol was removed by filtration and then freeze-dried to obtain 65 g of the bone resorption inhibitor of the present invention comprising DNA powder. Note that 1 g of this bone resorption inhibitor contained 425 mg of histone.

[Test Example 1]
β ₂ microglobulin, histone, complement third component, monocyte chemotactic protein 1, lysozyme, and ribonuclease were examined for bone resorption inhibitory action. Each active ingredient used for the test was a commercially available product. The origin of each active ingredient subjected to the test and the final concentration of each active ingredient are shown in Table 1.

After removing the long bones of 10-20 days old ICR mice and removing soft tissues, the bones are mechanically minced in α-MEM (Flow Laboratories) solution containing 5% fetal bovine serum. And whole bone marrow cells containing osteoclasts were obtained. The whole bone marrow cells containing osteoclasts were seeded on ivory pieces so as to be about 2 × 10 ⁶ cells, and spotted with an α-MEM solution containing 5% fetal bovine serum. After 2 hours, add β ₂ microglobulin, histone, complement third component, monocyte chemotactic protein 1, lysozyme, and ribonuclease to 5% fetal calf serum to the final concentrations shown in Table 1. Including α
-MEM solution was added and cultured for 5 days, and the bone resorption inhibitory activity of existing osteoclasts was examined.

Evaluation of the bone resorption inhibitory effect was done by detaching the cells on the ivory piece after staining and staining with hematoxylin,
Image analysis with PIASLA-555 was performed to determine the number of bone resorption pits (Seno Junji et al., Animal Culture Cell Manual by Research Theme, pp.199-200, 1993). That is, the small number of pits means that the activity of osteoclasts was reduced and the bone resorption activity was suppressed. In addition, substances that have been shown to inhibit bone resorption by pit assay have also been shown to inhibit bone resorption in animal experiments (Toba et al., Bone, vol.27, p.403-408, 2000). The pit assay is a suitable experimental system for examining the bone resorption inhibitory effect. As a control, the one with no active ingredient added was used, and the bone resorbing activity with no active ingredient added was defined as 100%.
%). The results are shown in Table 1.

本発明の有効成分であるβ₂ミクログロブリン、ヒストン、補体第３成分、単球走化性
タンパク質１、リゾチーム、及びリボヌクレアーゼを添加したものはいずれも、有効成分
無添加のものに比べ骨吸収活性が抑制されており、顕著な骨吸収抑制作用を有することが
判った。

In the present invention, β ₂ microglobulin, histone, complement third component, monocyte chemotactic protein 1, lysozyme, and ribonuclease added are all bone resorbed compared to those without any active ingredient added. It was found that the activity was suppressed and it had a remarkable bone resorption suppressing action.

After mixing the raw materials with the formulation shown in Table 2, the container was filled and sterilized by heating to produce a bone resorption suppressing beverage.

  After mixing the raw materials with the formulation shown in Table 3, it was pressure-molded to produce a bone resorption-suppressing tablet.

Raw materials were mixed with the composition shown in Table 4, and a dough was prepared and molded, and then baked to produce a bone resorption suppressing biscuit.

After mixing the raw materials with the formulation shown in Table 5, the container was filled and sterilized by heating to produce a bone resorption suppression jelly.

After mixing the raw materials with the formulation shown in Table 6, the processed cheese for inhibiting bone resorption was produced by emulsifying at 85 ° C.

After 12% by weight reduced skim milk was sterilized by heating at 90 ° C for 20 minutes, Lactobacillus acidophilus (L
actobacillus acidophilus) and Streptococcus thermophilus (Streptococcus)
thermophilus) was inoculated, and two types of starter cultures were obtained and mixed in equal amounts. And after mixing a raw material with the mixing | blending shown in Table 7, it was made to ferment and the yogurt for bone resorption suppression was manufactured.

Ingredients were mixed in the formulation shown in Table 8 to produce infant formula for inhibiting bone resorption.

Raw materials were mixed in the formulation shown in Table 9 to produce a dog breeding feed for suppressing bone resorption (dog food).

Claims (3)

Bone resorption inhibitor containing histone as an active ingredient. The bone resorption inhibitor according to claim 1, wherein 1 ng to 1 g is administered per day. A medicament for inhibiting bone resorption, comprising the bone resorption inhibitor according to claim 1 or 2.