JP2010254718A - Bone formation accelerator - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a bone formation accelerator etc., accelerating the growth of osteoblast and having an effect in treatment and/or prevention for the purpose of bone strengthening. <P>SOLUTION: This bone formation accelerator contains sphingophosphoglycolipid extracted from echinodermata as an effective component. This bone formation accelerator is disclosed as a composition for prevention and/or treatment of osteoporosis etc. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to an osteogenesis promoter that promotes the proliferation of osteoblasts and has an effect on treatment and / or prevention aimed at strengthening bone. More specifically, the present invention is purified from an organism belonging to the echinoderm. The present invention relates to a pharmaceutical osteogenesis promoter containing a sphingoline glycolipid as an active ingredient. In the present invention, the osteogenesis promoter means an osteogenesis promoter as a pharmaceutical composition. The present invention provides a novel osteogenesis promoter, and makes it possible to provide a food / beverage composition or a feed composition containing the above components.

  With aging, bone diseases such as osteoporosis, fractures, and back pain are increasing. Osteoblasts and osteoclasts are present in the bone tissue, and are responsible for bone formation and bone resorption, respectively, and so on. Known diseases caused by abnormal bone metabolism include osteoporosis, rheumatoid arthritis, Paget's disease of bone, and osteoarthritis. The problem of increasing bedridden elderly due to these bone diseases is As the society continues, it has become an issue that must be solved. Bone loss caused by abnormal bone metabolism is improved by promoting bone formation or suppressing bone resorption. The promotion of osteogenesis is the promotion of osteoblast proliferation / differentiation. In addition, suppression of bone resorption can be achieved by suppression of proliferation / differentiation of osteoclast precursor cells, function suppression of mature osteoclasts, and the like. The development of proteins and other compounds having such activities as promoting bone formation or inhibiting bone resorption is currently being promoted in various fields.

Osteoblasts are mononuclear cells derived from mesenchymal stem cells and are in a resting state that covers pre-osteoblasts, osteoblasts, and bone cells embedded in the bone matrix and bone surface Differentiate into cells. Proliferated and differentiated osteoblasts produce bone matrix (ostoids) and fill the bone resorption pits. Furthermore, under the control of osteoblasts, osteoids mature, mineralize into bone. Pre-osteoblasts and osteoblasts not only produce such bone matrix, but also insulin-like growth factors (IGF-1, IGF-2), β2-microglobulin, osteoinductive protein (BMP), transforming proliferation. Secretes various local factors such as factor-β (TGF-β), interleukin-1 (IL-1), tumor necrosis factor (TNF-α), prostaglandin E ₂ (PGE ₂ ), autocrine / The paracrine mechanism is thought to control bone formation. In addition, osteoblasts include parathyroid hormone (PTH), epidermal growth factor (EGF), active vitamin [1α, 25 (OH) ₂ D ₃ ], estrogen, TNF-α, PGE ₂ , IL-1, and the like. Therefore, it is considered that the control of proliferation / differentiation of osteoblasts and the differentiation / activation of osteoclasts are performed through these receptors.

Currently, the treatment of bone diseases, bisphosphonate compounds, calcitonin, active vitamin D ₃ formulations, hormone preparations of estradiol-containing pharmaceuticals such as vitamin K ₂ is generally used. In addition, active vitamin D ₃ derivatives, estradiol derivatives, third-generation bisphosphonate compounds, and the like have been developed with the aim of excellent therapeutic agents with few side effects. However, treatment methods for bone diseases using the aforementioned drugs are not always satisfactory, and there are some that are limited in terms of side effects. Furthermore, since multi-drug combination therapy has become the mainstream for the treatment of bone metabolic diseases, development of a new drug having an action mechanism different from that of conventional drugs is expected.

Sphingolipids exist widely in nature as important membrane components of cells, and most of them exist as glycosphingolipids and sphingophospholipids. Sphingolipids are known to play important roles in life phenomena such as regulatory effects on cell proliferation and differentiation (Non-patent Document 1) and apoptosis induction (Non-patent Document 2). It has been clarified that TNF (Non-patent Document 3), IL-1 (Non-patent Document 4) and Fas ligand (Non-patent Document 5), which are cytokines, are involved. Sphingolipids are involved in regulation of EGF receptor activation (Non-patent Document 6), intracellular fluidization of calcium ions (Non-patent Document 7), protein kinase C activity inhibition and activation, and the like. It is also known.
For glycosphingolipids, ceramide consisting of a long-chain base and a fatty acid is used as an aglycone, and cerebroside with a monosaccharide such as galactose or glucose bound to this aglycone, lactosylceramide with a lactose bound, sugar (monosaccharide or oligosaccharide) Sugar) and a sulfate group to which sulfatide is bonded, and ganglioside to which oligosaccharide and sialic acid are bonded are included (Non-patent Document 8). Among them, gangliosides containing sialic acid are used for mutual recognition (non-patent document 9), adhesion (non-patent document 10), proliferation (non-patent document 11), differentiation induction (non-patent document 12), and the like. It is said to be involved in various important phenomena in the body. This compound is also used for treatment of ischemic injury and brain injury caused by Parkinson's disease (Non-patent Document 13).

  The main skeleton of glycosphingolipids derived from organisms belonging to the marine invertebrates Echinodermata (for example, starfishes, spider starfishes, sea urchins, sea cucumbers, sea lilies, etc.) are mostly α-oxy fatty acids and sphingosine type Mammal origin that has a ceramide part (aglycone part) composed of long-chain bases or phytosphingosine-type long-chain bases, and is composed of fatty acids that are not oxidized at the α-position (unsubstituted fatty acids) and sphingosine-type long-chain bases It is structurally different from the glycosphingolipid (Non-Patent Document 8, Non-Patent Document 14). The phytosphingosine skeleton has a structure in which the double bond portion at the 4th and 5th positions in the sphingosine skeleton is saturated with a hydroxyl group at the 4th position and hydrogen at the 5th position. It is expected to have.

  Among the glycosphingolipids derived from echinoderms, glycolipids obtained from sea lilies (especially sea urchins) are sphingophosphoglycosides containing inositol phosphoceramide in the basic structure. This inositol phosphoceramide has been found in plants and anterior mouth animals, but it has only been found in posterior mouth animals from echinoderms. Unlike the conventional echinoderm glycosphingolipids, the ceramide part is composed of unsubstituted fatty acids and sphingosine-type long-chain bases, so the sphingoglycolipids of sea urchins are significantly different from those of other echinoderms. It is positioned as. As a technique using a ceramide having a phytosphingosine skeleton, a cosmetic composition (Patent Document 1), an external medicine for skin diseases (Patent Document 2), and the like are disclosed. In addition, a compound having a sphingosine skeleton such as ceramide, sphingomyelin, sphingoglycolipid, ganglioside and the like derived from bovine brain and milk, and further, one or more substances selected from calcium, vitamin D and vitamin K An added preventive and ameliorating agent for bone and joint diseases is disclosed (Patent Document 3).

JP-A-8-502961 JP 2001-39859 A JP 2001-158736 A

FEBS Lett., 524, 103-106, 2002 FEBS Letter, Vol. 526, pp. 15-20, 2002 EM BJ Journal (EMBO J.), Vol. 19, No. 13, pp. 3304-3313, 2000 Journal of Biological Chemistry (J. Biol. Chem.), 275, 45, 35617-35623, 2000 Journal of Biological Chemistry (J. Biol. Chem.), 276, 26, 23954-23961, 2001 Journal of Biological Chemistry (J. Biol. Chem.), 277, No. 12, pp. 10108-10113, 2002 Proceding National Academy of Sciences USA (Proc. Natl. Acad. Sci. USA), Vol. 98, No. 1, pp. 307-312, 2001 Nobuhiro Fushiya and Hiroshi Hamada, “Structural analysis of natural organic compounds”, Springer Fairlark Tokyo, pages 81-140, 1994 Journal of Biological Chemistry (J. Biol. Chem.), 264, 20159-20162, 1989 Journal of Biological Chemistry (J. Biol. Chem.), 266, 17552-17558, 1991 Brain Res., 284, 215-221, 1983 Proceding National Academy of Sciences USA (Proc. Natl. Acad. Sci. USA), Vol. 76, 3367-3371, 1979 Neurology, 50, 1630-1636, 1998 Pharmacia, Vol. 38, No. 9, pp. 851-855, 2002

  Various drugs are used to treat osteoporosis, one of the bone metabolic diseases, but it is necessary to grasp the patient's bone metabolic status when selecting a therapeutic drug, and bone turnover is highly metabolized. The applicable drug is determined depending on whether it is a rotational type or a low-turnover type. In the high turnover type, a bone resorption inhibitor that inhibits the effect of osteoclastic bone resorption is used, and in the low turnover type, an osteogenesis promoter that increases osteoblasts is used (Toshio Matsumoto, “Molecules” Bone Metabolism and Osteoporosis ”, Medical View, 293-295, 1996: These may be abbreviated as Prior Art 1). The compound having a sphingosine skeleton described in Patent Document 3 is a compound inherent to mammals, and inhibits bone resorption by osteoclasts, thereby exhibiting an effect of promoting the increase in bone density. Met.

  However, according to other prior art documents, osteoclasts and osteoblasts secrete local factors with each other and affect their differentiation and activation. Describes that it is considered highly likely to cause inactivation of osteoblasts and slow down bone turnover (Japanese Patent Laid-Open No. 10-29950, column 2, line 18). As is clear from this, it has been awaited in the technical field to develop a novel osteogenesis promoter useful for osteoporosis and other bone metabolic diseases.

The present inventors are considering the effective use of starfish belonging to the Echinodermata, which is feared to destroy the ecosystem of the fishing ground, using sharks as a diet, and being captured and discarded in large quantities. The α-oxy fatty acid and sphingoglycolipid having sphingosine skeleton or phytosphingosine skeleton extracted and purified from these, and sphingoline glycolipid have been found to have an effect of promoting osteoblast proliferation, and the present invention was completed. It came to do.
An object of the present invention is to provide a novel pharmaceutical osteogenesis promoter useful for the treatment and / or prevention of osteoporosis and other bone metabolic diseases.
On the other hand, the present invention is an echinoderm that is regarded as a serious environmental problem in the field of fisheries and has not yet been found to be effective even though it is captured in large quantities in the coastal areas of Japan. Providing a technology that contributes to the development of the fishery industry as well as solves environmental problems by producing food and beverage compositions and feed compositions to which the above ingredients having an effect of promoting bone formation are used. It is possible.

The present invention for solving the above-mentioned problems is a pharmaceutical osteogenesis promoter comprising a sphingoline glycolipid purified from an organism belonging to the echinodermata as an active ingredient. In the present invention, it is desirable that the organism belonging to the Echinodermata is an organism belonging to the sea lily net. Moreover, this invention makes it a desirable aspect that sphingoline glycolipid is inositol phosphoceramide and / or sialosyl inositol phosphoceramide.
Furthermore, the present invention has a desirable embodiment in which inositol phosphoceramide is inositol phosphoceramide of the following chemical formula 5.
15
Ins1 → Phospho-Cer
(Ins: Inositol)
However, R < ₃ > -Phospho-Cer shows the structural formula represented by Chemical formula 3 mentioned later.
Furthermore, this invention makes it a desirable aspect that the sialosyl inositol phosphoceramide is 1 type or multiple types of mixtures selected from the following sialosyl inositol phosphoceramide of Chemical formula 16-17.
16
9-OCH ₃ NeuGcα2 → 3Ins1 → Phospho-Cer
(NeuGc: N-glycolylneuraminic acid, Ins: inositol)
17
9-OCH ₃ NeuGcα2 → 11 [9-OCH ₃ ] NeuGcα2 → 3Ins1 → Phospho-Cer
(NeuGc: N-glycolylneuraminic acid, Ins: inositol)
However, R < ₃ > -Phospho-Cer shows the structural formula represented by Chemical formula 3 mentioned later.
Furthermore, the present invention also provides a desirable embodiment in which the osteogenesis promoter is a pharmaceutical composition for preventing / treating osteoporosis, rheumatoid arthritis, Paget's disease of bone, or osteoarthritis.

Next, the present invention will be described in more detail. In the present specification, percentages are expressed by mass unless otherwise specified.
The osteogenesis promoter of the present invention can be used for treatment and / or prevention aimed at bone strengthening such as treatment of bone metabolic diseases by promoting the proliferation of osteoblasts. It is considered useful as a therapeutic agent for rheumatoid arthritis, Paget's disease of bone, and osteoarthritis. Therefore, the osteoblast proliferation-promoting effect of the osteogenesis promoter of the present invention is an action located in the opposite relationship in bone metabolism to the effect of suppressing bone resorption by osteoclasts described in Patent Document 3. This is an effect that is not expected from the technique disclosed in Patent Document 3.

  The active ingredient of the osteogenesis promoter of the present invention is purified from an organism belonging to the echinoderm. Here, the organisms belonging to the Echinodermata are classified, specifically, the star class (eg, starfish, blue starfish, starfish starfish, starfish, purple starfish), snake starfish (eg, starfish) , Sea spider starfish, Japanese spider starfish, etc.), sea gall (e.g., sea urchin, bafun sea urchin, etc.), ridge (e.g., sea cucumber, e.g., sea cucumber), (Nippon sea fern, core sea fern, great sea fern etc.).

  In general, glycosphingolipids, as shown in FIG. 1, are ceramides consisting of a long-chain base having a sphingosine skeleton or a phytosphingosine skeleton and a fatty acid as aglycone, and in this aglycon part, neutral galactose, glucose or the like Cerebrosides with sugars, lactosylceramide with lactose, sulfatides with monosaccharides or oligosaccharides and sulfate groups, and neutral sugars and amino sugars (may not be included) and sial Those with acid binding are classified as gangliosides, etc. Incidentally, in glycosphingolipids derived from organisms belonging to the marine invertebrate Echinodermata, there are compounds in which the α-position of the fatty acid moiety shown in FIG. 1 is oxidized.

In general, as glycosphingolipid, specifically, ceramide, cerebroside, lactosylceramide, sulfatide, ganglioside, etc. having a sphingosine skeleton in which the α-position of fatty acid in the molecule is oxidized, or intramolecular The α-position of the fatty acid is oxidized to have a phytosphingosine skeleton (that is, a structure in which a hydroxyl group is saturated at the 4-position of the 4-position and 5-position double bond in the sphingosine skeleton, and hydrogen is saturated at the 5-position). Examples include ceramide, cerebroside, lactosylceramide, sulfatide, and ganglioside. The basic structure (R ₂ -Cer1) of ceramide having an α-oxy fatty acid and a sphingosine skeleton is shown in Chemical Formula 1, and the basic structure of ceramide having an α-oxy fatty acid and a phytosphingosine skeleton (R ₂ -Cer2) is shown in Chemical Formula 2, respectively. The active ingredient of the osteogenesis promoter of the present invention is a sphingoglycolipid that is a sphingoglycolipid containing inositol phosphoceramide in the basic structure. Specifically, as the sphingophosphoglycolipid, specifically, inositol phospholipid is used. Examples include ceramide or sialosylinositol phosphoceramide in which sialic acid or the like is bound thereto. The basic structure (R ₃ -Phospho-Cer) of inositol phosphoceramide is shown in Chemical formula 3. In the present specification, except for the case where special classification or explanation is made, the above-mentioned glycosphingolipid having an α-oxy fatty acid and a sphingosine skeleton, a glycosphingolipid having an α-oxy fatty acid and a phytosphingosine skeleton, and Glycosphingolipids are collectively referred to as glycosphingolipids.

  Here, the “active ingredient” means an ingredient having an effect of proliferating osteoblasts such as mammals in the osteogenesis promoter of the present invention, wherein the glycosphingolipid is the osteogenesis promoter of the present invention. It need not be the main component. Further, the osteogenesis promoter of the present invention may contain an ingredient having an osteoblast proliferation effect or an osteogenesis promoting effect in addition to the glycosphingolipid. The active ingredient of the osteogenesis promoter of the present invention can be produced by extraction and purification from echinoderms. Examples of the ingredient include an extract obtained from echinoderms as a raw material for extraction, and dilution of the extract. A liquid or a concentrated liquid, a dried product obtained by drying the extract, or a roughly purified product or a purified product thereof is used. Specific examples of echinoderms include marine starfish (for example, starfish, blue starfish, starfish starfish, starfish, purple starfish), serpentine starfish (eg, starfish, Japanese spider starfish), sea gall [ Sea urchins (for example, sea urchins, sea urchins, etc.), sea bream (for sea cucumbers) (for example, black sea cucumbers, blue sea cucumbers, etc.) From these, the active ingredient can be extracted and purified.

  As an example of the method for producing the active ingredient of the osteogenesis promoter of the present invention, a method based on the flowchart shown in FIG. 2 is preferably exemplified, and the specific contents thereof are as described below. That is, echinoderms are homogenized with a mixer, then extracted with a mixed solution of chloroform and methanol, filtered, and then the extract is concentrated. After adding water and suspending it, a mixed solution of ethyl acetate and n-butanol was added to perform a partitioning operation, and fractionated into an ethyl acetate / n-butanol layer (α) and an aqueous layer (β). Continue the extraction operation separately. The ethyl acetate / n-butanol layer (α) is concentrated, then acetone is added and mixed, and fractionated into an acetone-soluble fraction and an acetone-insoluble fraction. Next, the organic solvent contained in each fraction is volatilized to prepare extract A (acetone soluble fraction) and extract B (acetone insoluble fraction). Water-saturated n-butanol is added to the previously separated aqueous layer (β) to perform a partitioning operation, and the n-butanol layer (γ) and the aqueous layer (σ) are separated. The organic solvent in the n-butanol layer (γ) is volatilized to obtain extract C. Moreover, after concentrating a water layer ((sigma)), it melt | dissolves in 60% methanol. For separation of components, reverse phase column chromatography is performed by a stepwise gradient method in which the concentration of the methanol solution, which is the eluent, is increased stepwise to 60 to 100% and eluted, and the organic solvent of the 100% methanol elution fraction is removed. Volatilize to prepare extract D. Each prepared extract is then subjected to a separation step by normal phase column chromatography, etc., so that extract A to ceramide, extract B to cerebroside, extract C to lactosylceramide, and extract D to sulfatide, ganglioside, sphingoline sugar Each lipid can be purified.

  The solvent used in the extraction and purification of the osteogenesis promoter of the present invention is not particularly limited, but it is preferable to use water, an organic solvent, or a mixture thereof. Water that can be used as an extraction solvent is water that has been subjected to various treatments (eg, purification, heating, sterilization, filtration, ion exchange, osmotic pressure adjustment, buffering, etc.), such as purified water, hot water, Ion exchange water, physiological saline, phosphate buffer, phosphate buffered saline and the like are also included. Organic solvents that can be used as the extraction solvent include esters such as ethyl acetate and butyl acetate, alcohols such as methanol, ethanol, butanol, and isopropyl alcohol, aliphatic hydrocarbons such as heptane, hexane, and isooctane, and ketones such as acetone and methyl ethyl ketone. , Aromatic compounds such as benzene and toluene, ethers such as diethyl ether and t-butyl ether, lower aliphatic hydrocarbon compounds such as methylene chloride, chloroform and 1,2-dichloroethane, etc., among which methanol, n-butanol It is preferable to use chloroform. The concentration can be adjusted by appropriately selecting an organic solvent according to the kind of raw echinoderms used for production and the kind of active ingredient to be produced (sphingoglycolipid). In addition, several organic solvents can be mixed and used, and the mixing ratio and the like can be appropriately adjusted.

  The separation method used in the method for producing the active ingredient of the osteogenesis promoter of the present invention is preferably adsorption chromatography, partition chromatography, thin layer chromatography, high performance liquid chromatography (HPLC). Examples include a method of using one or a combination of gel-filled column chromatography and the like. The active ingredient of the osteogenesis promoter of the present invention is obtained from echinoderms by the above production method. The form of the composition of the present invention is not particularly limited, and examples of the form of the osteogenesis promoter of the present invention include an appropriate form as a pharmaceutical composition. The composition of the present invention may be a glycosphingolipid itself extracted and purified from echinoderms, or may contain components other than the glycosphingolipid. Components other than the glycosphingolipid can be appropriately selected according to the form of the composition.

  The pharmaceutical composition can be produced, for example, by formulating a glycosphingolipid using a pharmaceutically acceptable excipient or any other additive, and the formulated glycosphingolipid promotes bone formation. It can be used as an agent. In the case of formulation, the content of glycosphingolipid in the formulation is usually 0.000001 to 0.1% by mass, preferably 0.0001 to 0.01% by mass. In formulating, additives such as excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, diluents, and solvents for injection can be used. Examples of excipients include sugar derivatives such as lactose, glucose, mannitol and sorbit; starch derivatives such as corn starch, potato starch, α-starch, dextrin and carboxymethyl starch; crystalline cellulose, hydroxypropylcellulose, hydroxypropyl Cellulose derivatives such as methylcellulose, carboxymethylcellulose, carboxymethylcellulose calcium; gum arabic; dextran; pullulan; silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, magnesium magnesium aluminosilicate; phosphorus such as sodium phosphate, calcium phosphate Acid salt derivatives; carbonate derivatives such as sodium carbonate and calcium carbonate; and sulfate derivatives such as sodium sulfate and calcium sulfate.

  Examples of the binder include gelatin, polyvinyl pyrrolidone, macrogol and the like in addition to the above excipients. Examples of the disintegrant include, in addition to the above excipients, chemically modified starch or cellulose derivatives such as croscarmellose sodium, carboxymethyl starch sodium, and crosslinked polyvinylpyrrolidone. Examples of the lubricant include talc; stearic acid; stearic acid metal salts such as calcium stearate and magnesium stearate; colloidal silica; waxes such as bee gum and geirow; boric acid; glycol; carvone such as fumaric acid and adipic acid. Acids; Sodium carboxylic acid salts such as sodium benzoate; Sulfates such as sodium sulfate; Leucine; Lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; Silicic acids such as silicic anhydride and silicic acid hydrate; Starch derivatives Etc. Examples of the stabilizer include paraoxybenzoates such as methyl paraben and propyl paraben; alcohols such as chlorobutanol, benzyl alcohol and phenylethyl alcohol; benzalkonium chloride; acetic anhydride; sorbic acid and the like. Examples of the flavoring agent include sweeteners, acidulants, and fragrances. Examples of the solvent for injection include water, ethanol, glycerin and the like.

  Examples of the administration route of the pharmaceutical composition include parenteral administration such as oral administration and enteral administration. Examples of the dosage form of the pharmaceutical composition include injections, sprays, capsules, tablets, granules, syrups, emulsions, suppositories, ointments, tapes and the like. In addition, the pharmaceutical composition can be administered by being blended with food or drink or feed. The dose and frequency of administration vary depending on the intended effect, administration method, treatment period, age, sex, body weight, etc., but the dose is usually 0.001 to 1000 mg, preferably 0.1 to 0.1 mg per adult day. It can be appropriately selected from the range of 100 mg, and the number of administrations can be appropriately selected from the range of once to several times a day.

  Here, the food and drink composition shown as a reference example includes, for example, a glycosphingolipid which is an active ingredient of the bone formation promoter of the present invention, saccharides such as dextrin and starch; gelatin, casein, whey, soy protein, corn protein And the like; amino acids such as alanine, glutamine, and isoleucine; polysaccharides such as cellulose, guar gum, gellan gum, and gum arabic; and fats and oils such as soybean oil and medium chain fatty acid triglycerides. As a form of the food / beverage product composition, soft drinks, carbonated drinks, nutrition drinks, fruit juice drinks, lactic acid bacteria drinks and other drinks (including concentrated concentrates and powders for adjustment of these drinks); ice cream, sherbet, ice confectionery, etc. Noodles such as buckwheat, udon, harusame, dumplings and cucumber peels, Chinese noodles, instant noodles, etc .; confectionery such as candy, chewing gum, candy, chocolate, snack confectionery, biscuits, jelly, jam, cream, baked confectionery, tablet confectionery; Fish and fishery processed foods such as kamaboko, ham and sausage; Dairy products such as processed milk, fermented milk, and formula milk; salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, dressing and other fats and oils; Seasonings such as sauces, soups, soups, stews, salads, prepared dishes, pickles; breads; enteral nutrition; functions Food and the like. In addition, content of the sphingoglycolipid which is an active ingredient of the bone formation promoter added when manufacturing a food-drinks composition can be suitably selected from the range of 0.0001-0.1 mass%.

  The feed composition shown as a reference example includes, for example, glycosphingolipids, cereals such as corn, wheat, barley and rye; bran such as bran, wheat straw, rice bran and defatted rice bran; corn gluten meal and corn jam meal Such as non-fat dry milk, whey, fish meal, bone meal, etc .; yeasts such as beer yeast; mineral feeds such as calcium phosphate and calcium carbonate; fats and oils; amino acids; sugars, etc. Can be manufactured. Examples of the form of the feed composition include pet food (pet food, etc.), livestock feed, fish feed and the like. In addition, content of the sphingoglycolipid which is an active ingredient of the bone formation promoter added when manufacturing a feed composition can be suitably selected from the range of 0.0001-0.1 mass%. The pharmaceutical composition of the present invention may be used alone, but may be used in combination with other osteogenesis promoters, bone disease prevention / treatment agents, and the like. The combined use can enhance the effect of preventing and treating bone formation and bone disease. Further, the osteogenesis promoter and bone disease preventive / therapeutic agent to be used in combination may be contained as active ingredients in the composition of the present invention, or separate drugs without being contained in the composition of the present invention. Combined as a good product.

As described in detail above, the present invention relates to a pharmaceutical osteogenesis promoter containing a sphingoline glycolipid extracted from echinoderms as an active ingredient, and the present invention has the following effects. Is done.
(1) A novel osteogenesis promoter that promotes the proliferation of osteoblasts such as mammals can be provided.
(2) The osteogenesis promoter of the present invention is effective for the treatment and / or prevention of osteoporosis and other bone metabolic diseases.
(3) A pharmaceutical composition for prevention / treatment of osteoporosis and the like can be provided.
(4) It is possible to provide an effective utilization method of echinoderms captured in large quantities in the field of fisheries.

FIG. 1 shows the basic structure of glycosphingolipid. FIG. 2 is a flowchart showing a method for producing an α-oxy fatty acid and a sphingoglycolipid having a sphingosine skeleton or a phytosphingosine skeleton, and a sphingoline glycolipid from echinoderms. FIG. 3 shows the activity of promoting osteoblast proliferation by glycosphingolipid extracted and purified from echinoderms. FIG. 4 shows the activity of promoting osteoblast proliferation in osteoporosis model mouse bone marrow cells by glycosphingolipid extracted and purified from echinoderms.

Next, production examples of glycosphingolipids (ceramide, cerebroside, lactosylceramide, ganglioside) shown as reference examples and production examples of glycosphingolipid (sphingoglycolipid) which is an active ingredient of the bone formation promoter of the present invention explain. In addition, the structure of each glycosphingolipid was determined using a mass spectrum method (MS), a nuclear magnetic resonance spectrum method (NMR), etc.
Production Example 1
(Production of ceramide having α-oxy fatty acid and phytosphingosine skeleton)
1 kg of starfish starfish (scientific name: Luidia maculata) was homogenized with a mixer, extracted with 2 liters of a mixed solution of chloroform / methanol (chloroform: methanol = 1: 2), filtered, and the extract was concentrated. The concentrated fraction (41.4 g) was suspended by adding 250 ml of water, and then 250 ml of a mixed solution of ethyl acetate / n-butanol (ethyl acetate: n-butanol = 3: 1) was added to perform the partitioning operation. The ethyl acetate / n-butanol layer and the aqueous layer were separated. Of these, the ethyl acetate / n-butanol layer was concentrated, acetone was added to the concentrated fraction (9.9 g) and mixed, and the acetone-soluble fraction was recovered, and then the organic solvent was volatilized to extract about 5.3 g of Extract A. Got. Next, the extract A was dissolved in 5 ml of chloroform and added to a normal phase column (Silica gel 60: manufactured by Merck & Co., Inc.). A chloroform / methanol mixture (chloroform: methanol = 100: 0, 98: 2, 95) was used as an elution solvent. : 5, 9: 1, 85:15, 7: 3, and 6: 4), and the elution was performed by a stepwise gradient method in which the methanol concentration was increased stepwise. A fraction showing the same behavior as that of the existing ceramide was collected by normal phase thin layer chromatography, and the fraction was further added to a normal phase column and mixed with chloroform / methanol (chloroform: methanol = 97: 3 → 95: 5), and two types of ceramides, LMCer-1 (3 mg) and LMCer-2 (4 mg), each showing the behavior of a single substance by normal phase thin layer chromatography, were produced. The structural formula of LMCer-1 is shown in Chemical Formula 4, and the structural formula of LMCer-2 is shown in Chemical Formula 5, respectively.

4
H-Cer1
5
H-Cer2

Production Example 2
(Production of cerebroside having α-oxy fatty acid and phytosphingosine skeleton)
1 kg of starfish starfish was homogenized with a mixer, extracted with 2 liters of a mixed solution of chloroform / methanol (chloroform: methanol = 1: 2), filtered, and then concentrated. After adding 250 ml of water to this concentrated fraction and suspending it, 250 ml of a mixed solution of ethyl acetate / n-butanol (ethyl acetate: n-butanol = 3: 1) was added to carry out a partitioning operation. The n-butanol layer and the aqueous layer were separated. The ethyl acetate / n-butanol layer was concentrated and then added with acetone and mixed to collect an acetone-insoluble fraction, and the organic solvent was evaporated to obtain about 4.5 g of extract B. Next, the extract B is dissolved in 5 ml of a mixed solution of chloroform / methanol / water (mixing ratio = 9: 1: 0.05) and added to a normal phase column (Silica gel 60: manufactured by Merck). Eluted. The fraction showing the same behavior as that of the existing cerebroside was recovered by normal phase thin layer chromatography, and the fraction was added to a reverse phase column (Cosmosil 140 C18-PREP: manufactured by Nacalai Co., Ltd.) and added to 100% methanol. And eluted. Next, a fraction showing the same behavior as that of the existing cerebroside in normal phase thin layer chromatography was added to a gel filtration column (Sephadex LH-20: manufactured by Pharmacia), and chloroform / methanol (chloroform: methanol = 1). 1) Column chromatography using the mixed solution as an eluent, and two types of cerebrosides, LMC-1 (5 mg) and LMC-2 (6 mg), each showing the behavior of a single substance by normal phase thin layer chromatography, Manufactured. The structural formula of LMC-1 is shown in Chemical Formula 6, and the structural formula of LMC-2 is shown in Chemical Formula 7, respectively.

6
Glcβ1 → 1Cer1
(Glc: glucose)
7
Glcβ1 → 1Cer2
(Glc: glucose)

Production Example 3
(Production of lactosylceramide having α-oxy fatty acid and phytosphingosine skeleton)
1 kg of starfish starfish was homogenized with a mixer, extracted with 2 liters of a mixed solution of chloroform / methanol (chloroform: methanol = 1: 2), filtered, and then concentrated. After adding 250 ml of water to this and suspending, 250 ml of a mixed solution of ethyl acetate / n-butanol (ethyl acetate: n-butanol = 3: 1) was added to perform a partitioning operation, and ethyl acetate / n-butanol was added. Separated as a layer and an aqueous layer. Here, a water-saturated n-butanol is further added to the separated aqueous layer to perform a partitioning operation, and the n-butanol layer and the aqueous layer are separated, and the organic solvent is volatilized in the n-butanol layer and extracted. C. The dry weight of Extract C was about 3.2 g. Next, the extract C was dissolved in 5 ml of a mixed solution of chloroform / methanol (chloroform: methanol = 1: 1), added to a reverse phase column (Cosmosil 140 C18-PREP: manufactured by Nacalai), and eluted with the same mixed solution. . Subsequently, the fraction which showed the same behavior as the existing lactosylceramide was collect | recovered by normal phase thin layer chromatography. The fraction was added to a normal phase column (Silica gel 60: manufactured by Merck), and the mixing ratio was chloroform: methanol: water = 9: 1.5: 0.5, 8: 2: 0.2, and 6: Elution was performed by a stepwise gradient method using a 4: 1 each chloroform / methanol / water mixture and gradually increasing the methanol concentration. Among the eluted fractions, two types of lactosylceramide, LMCDH-1 (1.7 mg) and LMCDH-2 (1.4 mg), each showing the behavior of a single substance by normal phase thin layer chromatography, were produced. The structural formula of LMCDH-1 is shown in Chemical Formula 8, and the structural formula of LMCDH-2 is shown in Chemical Formula 9, respectively.

8
Galβ1 → 4Glcβ1 → 1Cer1
(Gal: galactose, Glc: glucose)
9
Galβ1 → 4Glcβ1 → 1Cer2
(Gal: galactose, Glc: glucose)

Production Example 4
(Production of ganglioside having α-oxy fatty acid and phytosphingosine skeleton)
1 kg of starfish starfish was homogenized with a mixer, extracted with 2 liters of a mixed solution of chloroform / methanol (chloroform: methanol = 1: 2), filtered, and then concentrated. After adding 250 ml of water to this and suspending, 250 ml of a mixed solution of ethyl acetate / n-butanol (ethyl acetate: n-butanol = 3: 1) was added to perform a partitioning operation, and ethyl acetate / n-butanol was added. Separated as a layer and an aqueous layer. Here, water-saturated n-butanol was further added to the separated water layer to perform a partitioning operation, and the n-butanol layer and the water layer were separated, and the water layer was recovered. After concentrating the aqueous layer, separation was performed using a reverse phase column (Cosmosil 140 C18-PREP: manufactured by Nacalai Co., Ltd.). Separation of the components was performed by a stepwise gradient method in which the concentration of the methanol solution as an eluent was gradually increased to 60 to 100% and eluted. About 0.18 g of extract D was prepared by volatilizing the organic solvent of the elution fraction when the concentration of methanol reached 100%. Next, the extract D is dissolved in a mixed solution of chloroform / methanol / water (chloroform: methanol: water = 6: 4: 1) and added to a normal phase column (Silica gel 60: manufactured by Merck). Eluted. Subsequently, sulfatide LMG-1 (0.67 mg) and ganglioside LMG which are added to a gel filtration column (Sephadex LH-20: manufactured by Pharmacia) and desalted, and exhibit the behavior of a single substance by normal phase thin layer chromatography. -2 (2.4 mg) was prepared respectively. The structural formula of LMG-1 is shown in Chemical Formula 10, and the structural formula of LMG-2 is shown in Chemical Formula 11, respectively.

10
3-O-HSO ₃ Galβ1 → 4Galβ1 → 4Glcβ1 → 1Cer2
(Gal: galactose, Glc: glucose)
11
NeuAcα2 → 3Galβ1 → 4Glcβ1 → 1Cer2
(NeuAc: N-acetylneuraminic acid, Gal: galactose, Glc: glucose)

Production Example 5
(Production of ganglioside having α-oxy fatty acid and phytosphingosine skeleton)
Ganglioside LLG-3 (58 mg) and LLG-5 (70 mg) were produced in the same manner as in Production Example 4, using 18 kg of Ao starfish (scientific name: Linkia laevigata) as a starting material. The structural formula of LLG-3 is shown in Chemical formula 12, and the structural formula of LLG-5 is shown in Chemical formula 13.

12
8-O-CH ₃ NeuAcα2 → 11 NeuGcα2 → 3Galβ1 → 4Glcβ1 → 1Cer2
(NeuAc: N-acetylneuraminic acid, NeuGc: N-glycolylneuraminic acid, Gal: galactose, Glc: glucose)
13
8-O-CH ₃ NeuGcα2 → 11NeuGcα2 → 11NeuGcα2 → 3Galβ1 → 4Glcβ1 → 1Cer2
(NeuGc: N-glycolylneuraminic acid, Gal: galactose, Glc: glucose)

Production Example 6
(Production of ganglioside having α-oxy fatty acid and phytosphingosine skeleton)
Ganglioside GAA-7 (86 mg) was produced in the same manner as in Production Example 4, using 65 kg of Murasaki starfish (scientific name: Asterias aurensis versicolor) as a starting material. The structural formula of GAA-7 is shown in Chemical formula 14.

14
8-O-CH ₃ NeuGcα2 → 6 [8-O-CH ₃ NeuGcα2 → 3] GalNAcβ1 → 3Galβ1 → 4Glcβ1 → 1Cer2
(NeuGc: N-glycolylneuraminic acid, GalNAc: N-acetylgalactosamine, Gal: galactose, Glc: glucose)

Production Example 7
(Manufacturing of sphingoline glycolipid)
Using 7.6 kg of Nipponumishima (scientific name: Mananthus japonica) as a starting material, an extract D derived from Nipponumishima was produced in the same manner as in Production Example 4. The extract D is dissolved in 2 ml of a chloroform / methanol / water mixture (chloroform: methanol: water = 8: 2: 0.2) and added to a normal phase column (Silica gel 60: manufactured by Merck). Were eluted by a stepwise gradient method using increasing chloroform concentration using chloroform: methanol: water = 8: 2: 0.2 and 6: 4: 1 chloroform / methanol / water mixtures. Sphingoline glycolipids CJP-1 (92 mg), CJP-2 (170.6 mg) and CJP-3 (568.5 mg) were prepared on thin layer chromatography. The structural formula of CJP-1 is shown in chemical formula 15, the structural formula of CJP-2 is chemical formula 16, and the structural formula of CJP-3 is shown in chemical formula 17.

15
Ins1 → Phospho-Cer
(Ins: Inositol)
16
9-OCH ₃ NeuGcα2 → 3Ins1 → Phospho-Cer
(NeuGc: N-glycolylneuraminic acid, Ins: inositol)
17
9-OCH ₃ NeuGcα2 → 11 [9-OCH ₃ ] NeuGcα2 → 3Ins1 → Phospho-Cer
(NeuGc: N-glycolylneuraminic acid, Ins: inositol)

  In the production examples 1 to 6, the α-oxy fatty acid and the sphingoglycolipid having a sphingosine skeleton or phytosphingosine skeleton extracted and purified from the echinoderms by the present inventors are purified from mammals and plants. Unlike the compound having a sphingosine skeleton described in Patent Document 3, the compound is confirmed to have a structure specific to echinoderms and the α-position of the fatty acid in the molecule is oxidized. I understood that. Moreover, the sphingoline glycolipid produced in Production Example 7 had a sphingophosphoglycolipid structure unique to sea lilies and sea urchins.

Next, a test example is shown and this invention is demonstrated in detail.
Test example 1
This test was conducted to measure the osteoblast proliferation promoting activity of glycosphingolipid extracted and purified from echinoderms.
(1) Preparation of sample Each glycosphingolipid produced in Production Examples 1 to 7 (LMCer-1, LMCer-2, LMC-1, LMC-2, LMCDH-1, LMCDH-2, LLG-3, LLG- 5, GAA-7, CJP-1, CJP-2, CJP-3) were used as test samples, respectively. Moreover, C2 ceramide (N-Acetyl-D-sphingosine synthetic: catalog No. A7191) which is a kind of conventional glycosphingolipid having no α-oxy fatty acid and phytosphingosine skeleton was used as a control sample.

(2) Test method 5 × 10 ⁴ mouse osteoblast cell line MC3T3-E1 (RCB1126: Riken Cell Bank) in each α-MEM medium (manufactured by Gibco BRL) containing 10% fetal bovine serum The resulting solution was dispensed into a 96-well microplate and cultured at 37 ° C. in the presence of 5% carbon dioxide gas for 18 hours. After culturing, the medium was removed, and 100 μl of α-MEM medium containing 2% fetal calf serum was added to each well. Then, each test sample and control sample were added to each well so that the final concentration was 0.2 μg / ml, and further cultured for 2 days. Tritium thymidine was added to each well at an amount of 50 nCi 6 hours before the end of the culture. After completion of the culture, the amount of DNA synthesized in each well was measured using a liquid scintillation counter (LKB1219 RACK-BETA: manufactured by LKB).

(3) Test results The results of this test are shown in FIG. FIG. 3 shows the activity of promoting osteoblast proliferation by glycosphingolipid extracted and purified from echinoderms. As a result, it was revealed that the glycosphingolipid derived from echinoderms used in each test sample has a high osteoblast proliferation promoting effect on C2 ceramide as a control sample. Among them, glycosphingolipids having a phytosphingosine skeleton (LMer-2, LMC-2, LMCDH-2, LLG-3, LLG-5, GAA-7) are generally more active than those having a sphingosine skeleton. There was found. Therefore, glycosphingolipids having a structure specific to echinoderms (for example, having an α-oxy fatty acid, a phytosphingosine skeleton, etc.) have a stronger osteoblast proliferation promoting effect than conventional glycosphingolipids. It became clear.

Test example 2
This test was performed to measure the ex vivo osteoblast proliferation-promoting activity of mice administered with glycosphingolipid extracted and purified from echinoderms.
(1) Preparation of sample Among the glycosphingolipids produced in the above production examples, LMMER-2, LMCDH-2, CJP-3, and LGG-3 were used as test samples, respectively. Moreover, physiological saline was used as a negative sample.

(2) Test method In this test, a 4-week-old female ddy mouse (purchased from Japan SLC) was used as the mouse. After the ovaries of the mice were removed, osteoporosis model mice prepared by feeding a low calcium diet for 7 weeks were divided into 6 groups per group. Each test sample solution prepared to 10 μg / ml with physiological saline was intravenously administered to the osteoporosis model mouse, 200 μl each, from the tail vein once a day for 14 days. After the administration was completed, bone marrow cells of femur and tibia were collected in RPMI 1640 medium (Gibco BRL). The collected osteoblasts were dispensed into 6-well microplates at 1 × 10 ⁶ per well and cultured for 7 days in RPMI 1640 medium containing 10% fetal bovine serum. After completion of the culture, alkaline phosphatase staining (DIAGNOSTICS, No. 85: manufactured by Sigma) was performed, and positive colonies were counted as osteoblast colonies.

(3) Test results The results of this test are shown in FIG. FIG. 4 shows the activity of promoting osteoblast proliferation in osteoporosis model mouse bone marrow cells by glycosphingolipid extracted and purified from echinoderms. As a result, it was found that any of the test samples had an action of proliferating osteoblasts in vivo.

Test example 3
This test was performed to measure the ex vivo osteoblast proliferation promoting activity of mice administered with a sphingoglycolipid extract, an intermediate purified product from echinoderms.
(1) Preparation of Sample Extract A (ceramide composition) roughly purified in Production Example 1; Extract B (Cerebroside composition) roughly purified in Production Example 2; Extract C (Lactosylceramide) roughly purified in Production Example 3 Composition) and extract D (ganglioside composition) roughly purified in Production Example 4 were used as test samples, dissolved in physiological saline to 500 μg / ml, and used for the test. At this time, Extract A contains 1 μg of ceramide, Extract B contains 1.2 μg of cerebroside, Extract C contains 0.5 μg of lactosylceramide, and Extract D contains 8 μg of ganglioside. Further, a solution prepared by preparing a ceramide derived from a mammal (manufactured by Wako Pure Chemical Industries, Ltd., catalog No. 031-18441) to 20 μg / ml with physiological saline was used as a control sample A, and cerebroside (manufactured by Wako Pure Chemical Industries, Ltd., catalog No. 0.076-03963) with physiological saline to 20 μg / ml was used as control sample B, and a ganglioside mixture (manufactured by Wako Pure Chemical Industries, Ltd., catalog No. 074-03643) was prepared with physiological saline to 20 μg / ml. The obtained solution was used as a control sample D, and physiological saline was used as a negative sample for each test.

(2) Test method In this test, as a mouse, a 6-week-old male db / db mouse (purchased from CLEA Japan) was used. The mice were grouped into 5 mice per group, and 500 μl each of extract A, extract B, extract C, extract D, control sample A, control sample B, control sample D, and negative sample were once a day. Forcible oral administration was continued for 36 days using a sonde. After the administration was completed, bone marrow cells of femur and tibia were collected in RPMI 1640 medium (Gibco BRL). The collected osteoblasts were dispensed into 6-well microplates at 1 × 10 ⁶ per well and cultured for 7 days in RPMI 1640 medium containing 10% fetal bovine serum. After completion of the culture, alkaline phosphatase staining (DIAGNOSTICS, No. 85: manufactured by Sigma) was performed, positive colonies were counted as osteoblast colonies, and the average value of 5 mice per group was calculated.

(3) Test results Table 1 shows the results of this test. Table 1 shows osteoblast proliferation promoting activity by glycosphingolipid extract, which is an intermediate purified product from echinoderms. As a result, the extract A, the extract B, the extract C, the extract D, the control sample A, the control sample B, and the control sample D all have an increased osteoblast colony count and a high growth promoting activity with respect to the negative sample. It has been found. However, it is negative for glycosphingolipid extract of the present invention (extract A, extract B, extract C, extract D), commercially available ceramide (control sample A), cerebroside (control sample B), and ganglioside mixture (control sample D). When the percentage of increase in the number of osteoblast colonies relative to the sample was compared, the extract A (ceramide composition): 5.0 compared to the control sample A: 3.2, and the extract B (cerebroside) compared to the control sample B: 1.1. Composition): 2.4, Control sample D: Extract D (ganglioside composition): 7.6 compared to 1.3, and therefore, the echinoderm-derived glycosphingolipid extract is derived from a conventional mammal. It was found to have an excellent osteoblast proliferation promoting effect as compared with glycosphingolipids.

Next, reference examples and reference examples are shown.
Example 1 (Reference Example)
An injection having the following composition was produced by a conventional method.
Ceramide (LMCer-2) produced in Production Example 1 0.01 (%)
Actinomycin D (manufactured by Sigma) 0.005
Sodium chloride (Wako Pure Chemical Industries, Ltd.) 0.9
Mannitol (manufactured by Kanto Chemical Co., Inc.) 1.0
Distilled water for injection (Otsuka Pharmaceutical Co., Ltd.) 98.085

Reference example 1
10.8 kg of whey protein enzyme degradation product (manufactured by Morinaga Milk Industry Co., Ltd.), 36 kg of dextrin (manufactured by Showa Sangyo Co., Ltd.), and a small amount of water-soluble vitamins and minerals were dissolved in 200 kg of water, and an aqueous phase was prepared in the tank. Separately, soybean salad oil (manufactured by Taiyo Yushi Co., Ltd.) 3 kg, palm oil (manufactured by Taiyo Yushi Co., Ltd.) 8.5 kg, safflower oil (manufactured by Taiyo Yufu Co., Ltd.) 2.5 kg, lecithin (manufactured by Ajinomoto Co., Inc.) 0.2 kg, An oil phase was prepared by mixing and dissolving 0.2 kg of a fatty acid monoglyceride (manufactured by Kao Corporation) and a small amount of a fat-soluble vitamin. The oil phase was added to the aqueous phase in the tank, stirred and mixed, then heated to 70 ° C., and further homogenized with a homogenizer at a pressure of 14.7 MPa. Next, the mixture was sterilized at 90 ° C. for 10 minutes, concentrated and spray-dried to prepare about 59 kg of intermediate product powder. 50 kg of this intermediate product powder, 6.8 kg of sucrose (manufactured by Hokuren), 167 g of amino acid mixed powder (manufactured by Ajinomoto Co., Inc.), and sphingophosphoglycolipid (CJP-3) produced in Production Example 7 above
) 60 g was added and mixed uniformly to produce about 56 kg of enteral nutrition food powder containing sphingoline glycolipid and having osteogenesis promoting effect.

Reference example 2
100 g of ganglioside (GAA-7) produced in Production Example 6, 110 g of lactulose powder (manufactured by Morinaga Milk Industry), 655 g of maltextrin (manufactured by Matsutani Chemical Industry), 95 g of skim milk powder (manufactured by Morinaga Milk Industry), stevia sweetener ( Each powder of 1 g of Saneigen F.F.I.), 5 g of yogurt flavor (manufactured of Saneigen F.F.I.), and 34 g of glycerin fatty acid ester preparation (manufactured by Riken Vitamin Co., Ltd.) was added and mixed uniformly. Using a tableting machine (manufactured by Hata Steel Co., Ltd.), the mixed powder was continuously tableted at a tableting speed of 12 tablets / minute and a pressure of 9.8 KPa at a tablet weight of 0.5 g. 1800 tablets (about 900 g) containing ganglioside (GAA-7) were produced.

Reference example 3
10 kg of raw milk having a milk fat content of 3.5% and a non-fat milk solid content of 9.2% was homogenized and heat-sterilized at 90 to 92 ° C. for 10 minutes. After cooling to about 42 ° C., commercially available Streptococcus thermophilus and Lactobacillus Lactobacillus as starters
delbrueckii subsp. bulgaricus) was added to each 100 g of milk culture. The mixture was sufficiently stirred in the fermentation tank and allowed to stand at 42 to 45 ° C. for 4 hours for fermentation. The fermented milk was cooled to 5-8 ° C. with stirring, and then homogenized with a homogenizer to prepare homogenized fermented milk. Next, 60 g of pectin (manufactured by Saneigen FFI) and 30 g of cerebroside (LMC-2) produced in Production Example 2 were added to 5 kg of 17% granulated sugar (manufactured by Toyo Seika Co., Ltd.), and the temperature was 90 to 92 ° C. Was sterilized for 10 minutes and then cooled to 5 to 8 ° C. to prepare a sugar solution. All the homogenized fermented milk and sugar solution prepared by these methods were thoroughly mixed in a tank, filled in 120 ml each in a paper container, and sealed to produce drink yogurt.

  As described in detail above, the present invention relates to a pharmaceutical osteogenesis promoter containing a sphingoline glycolipid extracted from echinoderms as an active ingredient. According to the present invention, osteoblasts such as mammals are used. It is possible to provide a novel osteogenesis promoter that promotes the growth of the bone. The osteogenesis promoter of the present invention is effective in the treatment and / or prevention of osteoporosis and other bone metabolic diseases. INDUSTRIAL APPLICABILITY According to the present invention, a pharmaceutical composition for prevention / treatment of osteoporosis and the like can be provided, and an effective utilization method of echinoderms captured in large quantities in the field of fisheries industry can be provided.

Claims (6)

  A pharmaceutical osteogenesis promoter comprising a sphingoline glycolipid purified from an organism belonging to Echinodermata as an active ingredient.   The osteogenesis promoter according to claim 1, wherein the organism belonging to the Echinodermata is an organism belonging to the sea lily net.   The osteogenesis promoter according to claim 1 or 2, wherein the sphingoline glycolipid is inositol phosphoceramide and / or sialosylinositol phosphoceramide. The osteogenesis promoter according to claim 3, wherein the inositol phosphoceramide is inositol phosphoceramide of the following chemical formula 15.
15
Ins1 → Phospho-Cer
(Ins: Inositol)
However, R < ₃ > -Phospho-Cer shows the structural formula represented by the following Chemical formula 3.

Chemical 3

The osteogenesis promoter according to claim 3, wherein the sialosylinositol phosphoceramide is a mixture of one or a plurality of sialosylinositol phosphoceramides of the following chemical formulas 16 to 17.
16
9-OCH ₃ NeuGcα2 → 3Ins1 → Phospho-Cer
(NeuGc: N-glycolylneuraminic acid, Ins: inositol)

17
9-OCH ₃ NeuGcα2 → 11 [9-OCH ₃ ] NeuGcα2 → 3Ins1 → Phospho-Cer
(NeuGc: N-glycolylneuraminic acid, Ins: inositol)
However, R < ₃ > -Phospho-Cer shows the structural formula represented by the following Chemical formula 3.

Chemical 3

  The osteogenesis promoter according to any one of claims 1 to 5, which is a pharmaceutical composition for prevention / treatment of osteoporosis, rheumatoid arthritis, Paget's disease of bone, or osteoarthritis.

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