Osteoblast differentiation promoter and bone formation promoter

Abstract

To provide a novel agent having the effects of promoting differentiation of osteoblasts and promoting bone formation.SOLUTION: The present inventors found that matairesinol has a bone mineralization promoting effect using mouse preosteoblasts. These findings showed that matairesinol has an osteoblast differentiation promoting effect and a bone formation promoting effect. The present invention provides an osteoblast differentiation promoter comprising matairesinol as an active ingredient, and a bone formation promoter comprising the osteoblast differentiation promoter.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to an agent for promoting osteoblast differentiation and a bone formation promoter. [Background technology]

[0002] Bones are constantly being remodeled and this is maintained by a balance between bone resorption and bone formation. Osteoclasts break down old bone (bone resorption), and osteoblasts attach to the parts destroyed by osteoclasts to form new bone (bone formation). If the balance of this bone metabolism is disrupted, for example, if bone resorption exceeds bone formation, bone density decreases and bones become brittle.

[0003] Osteoporosis, for example, is known as one of the bone metabolic diseases. Osteoporosis is characterized by a decrease in bone mass and deterioration of the microarchitecture of bone tissue. Osteoporosis leads to increased bone fragility and fracture risk. Osteoporosis can affect not only postmenopausal women but also men, and it is known that the incidence of osteoporosis is rapidly increasing worldwide. Osteoporosis reduces the quality of life and requires high medical costs, so it is important to prevent osteoporosis.

[0004] One of the causes of decreased bone density is known to be the decrease in female hormones after menopause in women. It is also known that bone density can be decreased by a low-calcium diet, nutritional deficiencies due to dieting, lack of exercise, smoking, excessive alcohol consumption, certain diseases, and the effects of certain medications.

[0005] Osteoblasts form bone by producing bone matrix proteins on the bone surface and inducing mineralization. Many ingredients have been reported so far that promote osteoblast differentiation and thereby promote bone formation. For example, Patent Document 1 discloses a food composition for inducing osteoblast differentiation, which is characterized by containing at least one active ingredient selected from red oak and lollo rossa.

[0006] Furthermore, Patent Document 2 discloses an osteoblast differentiation promoting agent that contains an extract of cherry blossoms. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Special Publication No. 2022-101897 [Patent Document 2] Special Publication No. 2016-108301 Summary of the Invention [Problem to be solved by the invention]

[0008] As described above, promoting bone formation, which leads to the prevention, improvement, and treatment of bone metabolic diseases such as osteoporosis, is an important issue, and there is a demand for the development of new drugs that can promote osteoblast differentiation and promote bone formation.

[0009] Therefore, an object of the present invention is to provide a new drug that has the effect of promoting osteoblast differentiation and bone formation. [Means for solving the problem]

[0010] The present inventors have conducted extensive research to solve the above problems and found that matairesinol has an effect of promoting osteoblast differentiation. Matairesinol is a lignan that is abundantly contained in Forsythia sieboldii leaf extract. In addition to matairesinol, Forsythia sieboldii leaf extract contains arctigenin, a lignan, and acteoside, a polyphenol component. The present inventors used mouse precursor osteoblasts to examine the bone mineralization promoting effect of matairesinol, arctigenin, and acteoside. As a result, arctigenin and acteoside showed almost no effect of promoting osteoblast mineralization, while matairesinol showed a significant effect of promoting mineralization. These results strongly suggest that matairesinol has an effect of promoting osteoblast differentiation and has an effect of promoting bone formation. The present invention was completed based on these findings.

[0011] The present invention provides an agent for promoting osteoblast differentiation, which comprises matairesinol as an active ingredient.

[0012] The present invention also provides a bone formation promoter comprising matairesinol as an active ingredient. Effect of the Invention

[0013] The present invention can promote osteoblast differentiation and bone formation, and therefore can provide a novel drug useful for preventing, improving and treating bone metabolic diseases. [Brief description of the drawings]

[0014] [Figure 1] Graph showing the effect of matairesinol on mineralization of MC3T3-E1 cells. [Diagram 2] Graph showing the effect of arctigenin on calcification of MC3T3-E1 cells. [Diagram 3] Graph showing the effect of acteoside on calcification of MC3T3-E1 cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The present invention provides an agent for promoting osteoblast differentiation, which comprises matairesinol as an active ingredient.

[0016] Osteoblasts are differentiated from osteoprogenitor cells, which in turn are differentiated from mesenchymal stem cells. Osteoblasts are cells responsible for bone formation, and attach to areas destroyed by bone resorption to induce calcification. It is known that bone formation is promoted by promoting differentiation of mesenchymal stem cells into osteoblasts. In this specification, "differentiation of osteoblasts" refers to either or both of the differentiation of mesenchymal stem cells into osteoprogenitor cells and the differentiation of osteoprogenitor cells into osteoblasts.

[0017] In the present invention, "promoting" osteoblast differentiation includes, for example, increasing the amount or rate at which osteoblast precursor cells or mesenchymal stem cells differentiate into osteoblasts, as compared to when the promoter of the present invention is not ingested, and promoting calcification induced by osteoblasts.

[0018] Matairesinol is a lignan represented by the following formula, and is known to be abundant in, for example, Forsythia chinensis leaf extract. Matairesinol is also known to be contained in wheat, beans, nuts, vegetables, etc. [ka]

[0019] The matairesinol used in the present invention may be a commercially available product, may be extracted from tissues of higher plants by known methods, or may be chemically synthesized by known methods.

[0020] The present invention also provides an osteogenesis promoter comprising matairesinol as an active ingredient. The osteogenesis promoter of the present invention may contain the above-mentioned osteoblast differentiation promoter as matairesinol.

[0021] In the present invention, "osteogenesis" refers to the formation of new bone on the surface of a bone. In the present invention, "promoting" osteogenesis includes, for example, promoting calcification induced by osteoblasts, and increasing bone mass, bone volume fraction (BV / TV), bone density, and the like, compared to when the promoter of the present invention is not ingested.

[0022] The agent of the present invention can be any form of preparation.As an oral administration preparation, the agent of the present invention can be, for example, tablets such as sugar-coated tablets, buccal tablets, coated tablets and chewable tablets; troches; pills; powders; capsules including hard capsules and soft capsules; granules; and liquids such as suspensions, emulsions, syrups and elixirs.

[0023] The agent of the present invention may be a parenteral administration preparation such as intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection, transdermal administration, nasal administration, pulmonary administration, enteral administration, oral administration, and transmucosal administration. The agent of the present invention may be, for example, an injection, a transdermal absorption tape, an aerosol, or a suppository.

[0024] The agent of the present invention can also be provided as an external preparation. The external preparation of the present invention can be a medicine, a cosmetic, or the like. The external preparation of the present invention can be an external preparation for application to the skin, scalp, hair, mucous membrane, nails, or the like. The external preparation includes, for example, a cream, an ointment, a liquid, a gel, a lotion, an emulsion, an aerosol, a stick, a sheet mask, a solid, a foam, an oil, a tick, or the like application agent; a patch such as a cataplasm, a plaster, a tape, or a patch; and a spray.

[0025] The agent of the present invention can be used in medicine, cosmetics, food, etc. The agent of the present invention can further contain any component that is usually used in medicine, cosmetics, and food.For example, the agent of the present invention can further contain pharmaceutically acceptable base, carrier, excipient, binder, disintegrant, lubricant, colorant, etc.

[0026] Examples of the carrier and excipient used in the agent of the present invention include lactose, glucose, sucrose, mannitol, dextrin, potato starch, corn starch, calcium carbonate, calcium phosphate, calcium sulfate, and crystalline cellulose.

[0027] Examples of binders include starch, gelatin, syrup, tragacanth, polyvinyl alcohol, polyvinyl ether, polyvinylpyrrolidone, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, carboxymethyl cellulose, and the like.

[0028] Examples of disintegrants include starch, agar, powdered gelatin, crystalline cellulose, calcium carbonate, sodium bicarbonate, sodium alginate, sodium carboxymethylcellulose, and calcium carboxymethylcellulose.

[0029] Examples of the lubricant include magnesium stearate, hydrogenated vegetable oil, talc, macrogol, etc. As the coloring agent, any coloring agent permitted to be added to medicines, quasi-drugs, and foods can be used.

[0030] Furthermore, the agent of the present invention may be coated with one or more layers of white sugar, gelatin, purified shellac, gelatin, glycerin, sorbitol, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinylpyrrolidone, cellulose acetate phthalate, hydroxypropyl methyl cellulose phthalate, methyl methacrylate, methacrylic acid polymers, or the like, if necessary.

[0031] Furthermore, the agent of the present invention may contain, as necessary, pH regulators, buffers, stabilizers, preservatives, antiseptics, diluents, coating agents, sweeteners, flavors, solubilizers, and the like.

[0032] The agent of the present invention may also be a food for the sick, a health food, a food with functional claims, a food for specified health uses, a nutritional supplement, a supplement, or the like.

[0033] The content of matairesinol in the agent of the present invention may be an amount that can exert the effect of the present invention, and can be appropriately set according to the subject, purpose, and intake method (administration method) of application.For example, when orally ingested by humans, matairesinol can be contained so that the daily intake (dosage) is 5 mg or more, preferably 10 mg or more, more preferably 20 mg or more.In addition, the upper limit of the daily intake (dosage) of matairesinol is not particularly limited, but can be, for example, 1000 mg or less.

[0034] The agent of the present invention can be taken (administered) continuously, for example, for 2 days, 3 days, 5 days, 1 week, 2 weeks, or 3 weeks or more. The timing of taking (administering) the agent of the present invention is not particularly limited, but is preferably taken on an empty stomach, more preferably before or 2 to 3 hours after a meal, and even more preferably before or 2 to 3 hours after breakfast.

[0035] The agent of the present invention is not particularly limited and can be applied to any subject. Subjects to which the agent of the present invention is applied include, for example, humans and non-human animals, such as mammals such as mice, rats, rabbits, cats, dogs, cows, horses and monkeys. The agent of the present invention can also be applied to subjects for which promotion of bone formation is desired, such as subjects for which prevention, improvement and / or treatment of bone metabolism diseases such as osteoporosis is desired, without particular limitation. Bone metabolism diseases include, for example, postmenopausal osteoporosis, steroid-induced osteoporosis, osteoporosis caused by a low calcium diet, osteomalacia, rickets, Paget's disease of bone, and bone diseases caused by tumors.

[0036] As used herein, the term "treatment" includes inhibiting, alleviating or relieving a disease or condition, inhibiting, suppressing, slowing or eliminating the progression of a disease or condition, achieving remission, and achieving improved prognosis.

[0037] As used herein, the term "amelioration" includes alleviating or relieving a disease or symptom, as well as inhibiting, slowing or eliminating the progression of a disease or symptom.

[0038] As used herein, the term "prevention" includes inhibiting, suppressing or delaying the occurrence (development) of a disease or condition, and inhibiting, suppressing or delaying the recurrence of a disease or condition. EXAMPLES

[0039] (cell culture) Mouse MC3T3-E1 cells (ATCC, Manassas, Virginia, VA, USA) were used as osteoblast precursors. The cells were cultured in α-MEM medium containing 10% FBS, 100 units / ml penicillin, and 100 μg / ml streptomycin. When the cells reached 90% confluence, the medium was switched to mineralization medium containing 10 mM β-glycerol phosphate, 50 μg / mL ascorbic acid, and 10 nM dexamethasone.

[0040] (reagent) Matairesinol was purchased from Cayman Chemical Co. Acteoside was purchased from Tokyo Chemical Industry Co., Ltd.

[0041] Arctigenin used was a purified arctigenin product (>95.0%) purified from burdock sprout extract by Kracie Holdings, Inc.

[0042] (Alizarin Red S staining of osteoblasts) Four weeks after inducing osteoblast differentiation of MC3T3-E1 cells, the cells were fixed with 4% paraformaldehyde for 30 minutes at 25°C, and then incubated with 1% Alizarin Red S (Fujifilm) for 15 minutes at 25°C. After washing with distilled water, the staining solution was extracted with 5% formic acid, and the absorbance at 450 nm was measured.

[0043] (statistical analysis) All data were expressed as mean ± standard deviation (SD). Dunnett's test of one-way analysis of variance was used to compare each group with the control group as a multiple comparison method. P<0.05 was set as the significance level.

[0044] [Test Example 1: Bone mineralization promotion effect] The mineralization-promoting effects of matairesinol, arctigenin, and acteoside were investigated using mouse MC3T3-E1 osteoblast precursor cells. Acteoside and arctigenin were used as controls. Cells were cultured in mineralization medium for 3 weeks in the presence of different concentrations of each reagent, and stained with Alizarin Red S. The absorbance of the extracted staining solution was then measured.

[0045] The results of the absorbance measurements are shown in Figures 1 to 3 and Tables 1 to 3. Figure 1 and Table 1 show the effect of matairesinol on the calcification of MC3T3-E1 cells. Figure 2 and Table 2 show the effect of arctigenin on the calcification of MC3T3-E1 cells. Figure 3 and Table 3 show the effect of acteoside on the calcification of MC3T3-E1 cells. Data are expressed as the mean ± standard deviation (n = 3).

[0046] Arctigenin showed little effect in promoting calcification of osteoblasts. Acteoside showed a tendency to promote calcification at a concentration of 10 μM, but the promoting effect was not statistically significant. On the other hand, matairesinol showed a significant calcification promoting effect at concentrations of 0.1 μM, 1 μM, and 10 μM. These results strongly suggest that matairesinol has an effect of promoting osteoblast differentiation and promoting bone formation.

[0047] [Table 1]

[0048] [Table 2]

[0049] [Table 3] [Industrial Applicability]

[0050] The present invention can be suitably used in medicines, foods, and the like for promoting bone formation.

Claims

1. An osteoblast differentiation promoter containing matairesinol as an active ingredient.

2. A bone formation promoter containing matairesinol as an active ingredient.

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