KR102531782B1 - Osteoblast active composition containing lutonarin - Google Patents

Abstract

The present invention relates to an agent for preventing or treating bone disease, comprising lutonarin or a pharmaceutically acceptable salt thereof as an active ingredient. Since the compound of the present invention or a pharmaceutically acceptable salt or a pharmaceutically acceptable salt thereof exhibits an osteoblast activity effect, it can be used to develop bone disease treatment for preventing or treating the above bone disease.

Description

Composition for preventing or treating bone disease containing lutonarin {Osteoblast active composition containing lutonarin}

The present invention relates to a composition for preventing or treating bone disease containing rutonarin, and specifically, by confirming the osteoblast activity and osteoclast inhibitory effect of rutonarin or a pharmaceutically acceptable salt thereof, thereby preventing or treating bone disease thereof. It is to provide a composition, a food composition and a feed composition.

Bone supports the body's soft tissue and body weight and surrounds the internal organs to protect the internal organs from external shocks. In addition, it is one of the important parts of the human body that structurally supports muscles and organs and stores other essential minerals such as calcium, phosphorus or magnesium in the body.

Adult bones do not stop growing, and until the day of death, old bones are removed and new bones are replaced with new ones. The process of creation and absorption is very dynamic and continuously repeated, maintaining balance. This is called bone remodeling, and the bone cycle, which removes old bone and replaces it with new bone, is essential for restoring fine damage to bone caused by growth and stress and maintaining proper bone function. It is known that two types of cells are largely involved in the remodeling of the bone. One of the two types of cells is an osteoblast that produces bone, and the other is an osteoclast that destroys bone. .

The osteoblasts produce RANKL (Receptor activator of nuclear factor kappa-B ligand) and its induced receptor, Osteoprotegerin (OPG). When the RANKL binds to RANK, which is a receptor on the surface of osteoclast precursor cells, the osteoclast precursor cells mature into osteoclasts and bone resorption occurs. However, when osteoprotezerin (OPG) binds to RANKL, the binding between RANKL and RANK is blocked, so that the formation of osteoclasts is inhibited and bone resorption does not occur.

Osteoporosis, a typical bone-related disease, refers to a state in which bone strength is weakened due to a decrease in bone quantity and qualitative changes, resulting in a high possibility of fracture. Osteoporosis, which has been defined as a low bone mass (Mass) state, is one of the important social problems. In the case of the United States, about 260,000 women are induced every year, and about 12 to 24% of them die. In a situation where society is getting older and women's social participation is becoming more active, osteoporosis and fractures due to osteoporosis in the elderly or postmenopausal women cause serious problems.

The causes of low bone density are genetic factors, early menopause, drugs such as steroids, comorbid diseases, smoking and alcohol. etc. That is, it occurs in a state in which minerals (especially calcium) and substrates constituting bone are reduced, and the balance of bone remodeling is broken so that osteoclast action is increased more than osteoblast action. The inside of a normal bone forms a dense structure like a mesh, but in the case of osteoporosis, the gap between bone microstructures widens and the microstructure becomes thin and weak, increasing the risk of easy bone fracture even with a small impact.

The osteoporosis is a disease characterized by deterioration of the microarchitectural structure of bone tissue, which causes an increase in bone fragility and consequently an increase in the risk of fracture in the elderly. As such, osteoporosis is known to affect more than 50% of women over the age of 50 and more than 30% of men over the age of 50, and in women, the rate of bone loss accelerates immediately after menopause or in the years thereafter. Most are asymptomatic, but when a fracture occurs, pain occurs, and various symptoms may appear depending on the site of the fracture. Fractures can occur anywhere, but fractures are most common in the wrist, spine, and hip (femur). In addition, regardless of age, it can be classified as secondary osteoporosis caused by diseases, drugs, alcohol, smoking, or accidents.

Substances currently used for the treatment of osteoporosis include estrogen, androgenic anagolic steroids, bisphosphonates, parathyroid hormone, calcium preparations, phosphates, and fluoride, which are traditionally used in postmenopausal women. preparations, ipriflavone, and vitamin D3. Although the female hormone has the effect of reducing fractures, it should be used with caution because it increases the risk of thrombosis and breast cancer. Although it is a somewhat effective method for treating menopausal symptoms and maintaining bone density by reducing the survival period of women, it has side effects such as inducing endometrial hyperplasia. In order to compensate for these disadvantages, a selective estrogen receptor modulator (SERM) has been developed. Raloxifene, a drug in this class, is effective in preventing spinal fractures and has been reported to lower the incidence of breast cancer in some cases. In addition, the bisphosphonate preparation is the most widely used osteoporosis treatment, and reduces the function of osteoclasts and reduces their number to prevent bone destruction. In addition, strontium (Strontium ranelate) increases bone formation as an edible osteoporosis treatment, but there is a problem in that bone resorption is inhibited.

Therefore, there is a need for a composition made of natural materials capable of preventing, improving, and treating bone-related diseases and having no side effects.

Under this background, the present inventors determined that rutonarin or a pharmaceutically acceptable salt thereof would be used as a composition for preventing, improving or treating bone diseases, and as a result of diligent efforts to solve the conventional problems, rutonarin or a pharmaceutically acceptable salt thereof was found. The present invention was completed by confirming the osteoblast activity and osteoclast inhibitory effects of possible salts.

One object of the present invention is to provide a pharmaceutical composition for preventing or treating bone disease, comprising lutonarin or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention is to provide a method for preventing or treating bone disease comprising administering the pharmaceutical composition to a non-human subject.

Another object of the present invention is to provide a health functional food composition for preventing or treating bone disease, comprising lutonarin or a food-acceptable salt thereof as an active ingredient.

Another object of the present invention is to provide a feed composition for preventing or treating bone disease, comprising lutonarin or a food-acceptable salt thereof as an active ingredient.

A detailed description of this is as follows. Meanwhile, each description and embodiment disclosed in the present invention may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be seen that the scope of the present invention is limited by the specific descriptions described below.

A first aspect of the present invention for achieving the above object provides a pharmaceutical composition for preventing or treating bone disease, comprising lutonarin or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the second aspect of the present invention provides a method for preventing or treating bone disease comprising administering the pharmaceutical composition to a non-human subject.

In addition, the third aspect of the present invention provides a health functional food composition for preventing or treating bone disease, comprising lutonarin or a food chemically acceptable salt thereof as an active ingredient.

In addition, a fourth aspect of the present invention provides a feed composition for preventing or treating bone disease, comprising lutonarin or a food chemically acceptable salt thereof as an active ingredient.

Hereinafter, the present invention will be described in more detail.

The term of the present invention, "lutonarin" is a compound having the structure of Formula 1 below, and may be obtained from an extract containing the same, but may be isolated from barley or new barley, although not limited thereto. In addition, the rutonarin may be synthesized or purchased using a known method.

[Formula 1]

In addition, the rutonarin may include all derivatives within the range expected by those skilled in the art in rutonarin, and are included without limitation as long as they have the same effect in the present invention.

The term of the present invention, "osteoblast activity" is not particularly limited, but may act on osteoblasts as a bone formation stimulant to inhibit apoptosis and increase survival.

The term of the present invention, "osteoclast inhibition" is not particularly limited, but is formed through various steps including cell contact, association, and differentiation of osteoclasts mainly derived from the bone marrow hematopoietic mononuclear leukocyte/macrophage system. Osteoclast activation can lead to bone loss and consequently induced rheumatic joints and osteoporosis. Therefore, osteoclast inhibition may be to prevent various diseases related to bone loss, such as osteoporosis, by inhibiting the differentiation of osteoclasts.

The "bone disease" refers to osteoporosis, facet bone disease, rickets, osteomalacia, renal osteodystrophy in patients with renal failure, rheumatoid bone disease, degenerative bone disease, bone metastatic lesion, primary bone tumor, periodontal disease, inflammatory alveolar bone It may be selected from the group consisting of resorptive diseases and inflammatory bone resorption diseases.

The pharmaceutical composition comprising rutonarin or a pharmaceutically acceptable salt thereof of the present invention as an active ingredient is characterized in that it is for preventing or treating bone diseases.

In a specific embodiment of the present invention, it was confirmed that the rutonarin has the activity of the osteoblast activity marker ALP (alkaline phosphatase, basic phosphatase) and the osteoblast activity efficacy under BMP-2 treatment.

The term “bone morphogenetic protein (BMP)” of the present invention was found to have excellent osteoinductive ability among the regulators related to the differentiation of osteoblasts, and 15 types were found. This means that the purified protein itself shows the osteoinductive ability of the demineralized bone matrix.

In particular, in the case of BMP-2, when added to C2C12 cells, a mouse premyoblastic cell line in vitro, the differentiation of muscle cells is inhibited and it has the ability to transform into osteoblasts.

The term "ALP (alkaline phosphatase)" of the present invention is a basic phosphatase, and ALP present in the cell membrane of osteoblasts means a marker for osteoblast activity, and ALP activity is p-nitrophenyl phosphate (pNPP, Sigma-Aldrich) It can be calculated by measuring the amount of pnitrophenol produced when ALP acts as a catalyst for the hydrolysis reaction of , using a microplate reader at a wavelength of 405 nm. ALP acts as a transporter of inorganic phosphate and a regulator of cell division or differentiation during calcification. Therefore, it can be confirmed by measuring whether the sample increases ALP activity as a biomarker (biomarker) for determining the effect on the activity of osteoblasts.

Thus, it was confirmed that the pharmaceutical composition of the present invention can prevent or treat bone diseases, particularly osteoporosis, by activating osteoblasts with rutonarin or a pharmaceutically acceptable salt thereof.

As used herein, the term "pharmaceutically acceptable salt" refers to a salt in a form that can be used pharmaceutically among salts in which cations and anions are bonded by electrostatic attraction, and is usually combined with metal salts and organic bases. salts, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like. For example, the metal salt may be an alkali metal salt (sodium salt, potassium salt, etc.), an alkaline earth metal salt (calcium salt, magnesium salt, barium salt, etc.), aluminum salt and the like; Salts with organic bases include triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N-dibenzylethylenediamine It can be a salt with; Salts with inorganic acids may be salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and the like; Salts with organic acids include formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and the like; A salt with a basic amino acid may be a salt with arginine, lysine, ornithine, and the like; A salt with an acidic amino acid may be a salt with aspartic acid or glutamic acid.

As used herein, the term "prevention" refers to any activity that suppresses or delays the onset of bone disease by administration of the pharmaceutical composition according to the present invention.

As used herein, the term "treatment" refers to all activities that improve or beneficially change the symptoms of a subject suspected of having a bone disease or affected by the administration of the pharmaceutical composition.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent, and the carrier may include a non-naturally occurring carrier.

More specifically, carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, Calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, polycaprolactone, polylactic acid ( Poly Lactic Acid), poly-L-lactic acid, and mineral oil. The pharmaceutical composition may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions according to conventional methods, respectively, and the carrier In the form of may include various irregular carriers, microspheres, nanofibers, and the like. When formulated, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient such as starch, calcium carbonate, It may be prepared by mixing sucrose or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium styrate and talc may also be used. Liquid preparations for oral administration include suspensions, internal solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is. Formulations for parenteral administration may include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, suppositories, and the like. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. The content of rutonarin or a pharmaceutically acceptable salt thereof included in the pharmaceutical composition of the present invention is not particularly limited. Another embodiment of the present invention for achieving the above object provides a health functional food composition for preventing or improving bone disease comprising the rutonarin or a pharmaceutically acceptable salt thereof.

In another embodiment of the present invention for achieving the above object, since rutonarin according to the present invention exhibits an excellent ALP activity increasing effect, it can be included in a food composition for the purpose of preventing or improving bone disease, and the food composition is routinely Since it can be ingested, high effects can be expected for the prevention or improvement of bone diseases.

At this time, the description of prevention of "rutonarin", "bone disease", and "prevention" is the same as described above.

As used herein, the term "improvement" refers to all actions that at least reduce the parameters related to the condition to be treated by administering the health functional food composition according to the present invention, for example, the degree of symptoms.

The term of the present invention, "health functional food" refers to food manufactured and processed using raw materials or ingredients having useful functionality for the human body according to Act No. 6727 on Health Functional Food, and 'functional' refers to the structure of the human body. and to obtain useful effects for health uses such as regulating nutrients with respect to functions or physiological actions, and the like. On the other hand, health food refers to food that has an active health maintenance or promotion effect compared to general food, and health supplement food refers to food for the purpose of supplementing health. In some cases, the terms of health functional food, health food, and health supplement food can be mixed.

Rutonarin or a food chemically acceptable salt thereof of the present invention may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to conventional methods.

The food of the present invention can be prepared by a method commonly used in the art, and can be prepared by adding raw materials and components commonly added in the art during the preparation. Specifically, the food composition may further include a physiologically acceptable carrier, the type of carrier is not particularly limited, and any carrier commonly used in the art may be used. In addition, the food composition contains food additives such as preservatives, bactericides, antioxidants, colorants, coloring agents, bleaching agents, seasonings, sweeteners, flavoring agents, expanding agents, reinforcing agents, emulsifiers, thickeners, coating agents, gum base agents, foam inhibitors, solvents, and improvers. can include The additive may be selected according to the type of food and used in an appropriate amount.

In addition, the formulation of the food may be prepared without limitation as long as the formulation is recognized as food. The composition for food of the present invention can be prepared in various types of formulations, and unlike general drugs, it has the advantage of not having side effects that may occur when taking drugs for a long time using food as a raw material, and has excellent portability, so the present invention Of the foods can be consumed as supplements to enhance the effect of preventing or improving bone disease.

Rutonarin or a food chemically acceptable salt thereof according to the present invention may be included in a food composition in various weight percentages as long as it can prevent or improve bone diseases. Specifically, it may be included in 0.00001 to 100% by weight or 0.01 to 80% by weight relative to the total weight of the food composition, but is not limited thereto. In the case of long-term intake for health and hygiene purposes, the content below the above range may be included, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

Another embodiment of the present invention for achieving the above object provides a feed composition for preventing or improving bone disease comprising the rutonarin or a food chemically acceptable salt thereof. At this time, the descriptions of "rutonarin", "food acceptable salt", "bone disease", "prevention" and "improvement" are as described above.

Since rutonarin or a food chemically acceptable salt thereof according to the present invention exhibits excellent osteoblast activity, it can be included in a feed composition for the purpose of preventing or improving bone disease, and the feed composition is a food composition that animals routinely consume. Since it is possible, high effects can be expected for the prevention or improvement of bone diseases.

As used herein, the term "feed" refers to any natural or artificial diet, meal, etc., or component of said meal, intended for or suitable for consumption by animals. The type of feed is not particularly limited, and feeds commonly used in the art may be used. Non-limiting examples of the feed include vegetable feeds such as grains, root fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, meal or grain by-products; Animal feeds such as proteins, inorganic materials, oils, mineral oils, oils, single cell proteins, zooplankton, or food may be mentioned. These may be used alone or in combination of two or more.

In a specific example, rutonarin was confirmed to have an osteoblast activity effect and ALP activity under BMP-2 treatment.

Taken together, the present invention suggests that bone diseases can be prevented, improved or treated by activating osteoblasts using rutonarin.

Since rutonarin of the present invention exhibits osteoblast activity and osteoclast inhibitory effects, it can be used to develop a composition for preventing or treating osteoporosis.

1 is a photograph showing the osteoblast activity of rutonarin of the present invention as an ALP index.
Figure 2 is a graph showing the osteoblast activity of rutonarin (BS2).

Hereinafter, the present invention will be described in more detail through examples. These examples are intended to explain the present invention in more detail, and the scope of the present invention is not limited by these examples.

Example 1: Lutonarin extraction and fractionation

The barley was germinated after removing moisture after soaking, and after about 7-10 days, sprouted barley was harvested and freeze-dried. It was powdered to obtain sprouted barley powder. The obtained barley sprout powder was extracted by adding 50-80% alcohol, filtered to obtain a liquid component, and the liquid component was freeze-dried to obtain an extract of barley sprout. Rutonarin was isolated and purified from the obtained barley sprout extract. More specifically, 2L of purified water was added to 100g of barley sprout extract at room temperature, suspended, and fractionated in the order of n-hexane, ethyl acetate and butanol to obtain n-hexane fraction (12g), ethyl acetate fraction (15g) and butanol fraction (33g) got each. The obtained ethyl acetate and butanol fractions (48 g) were purified by reverse-phase C18 silica gel column chromatography (500 g, Merk) using sequentially water and methanol (mixing ratio of 20:1 to 1:10) mixed solvents as mobile phase solvents. Fractions (Fr1 to Fr4) were obtained. After Fr.2,3 (8.7 g) was adsorbed on reverse-phase C18 silica gel (250 g, Merk) again, column chromatography was performed. At this time, the mobile phase solvent obtained a pale yellow color using a methanol mixed solvent and purified Rutonarin (240mg) was obtained.

Example 2: confirmation of osteoblast differentiation promotion activity of rutonarin

In order to confirm the bone formation promoting effect of rutonarin obtained in Example 1, the osteoblast differentiation promoting effect was evaluated according to the treatment concentration (FIGS. 1 and 2).

Figure 1 is a photomicrograph showing the results of comparing the effect of rutonarin on osteoblast differentiation through ALP staining after osteoblast differentiation, Figure 2 is a photomicrograph showing the effect of rutonarin on osteoblast differentiation through ALP activity measurement after osteoblast differentiation This is a graph showing the results of comparing the effects of Narin.

Specifically, the osteoblast cell line C3H10T1/2 (Korean Cell Line Bank, Korea) was added with 10% fetal bovine serum (FBS) and 1% antibiotics (antibiotics; penicillin 100 U/ml and streptomycin 0.1 mg/ml) in DMEM. (Dulbecco's modified eagle medium) was used and cultured in a 96-well plate at 37° C., 5% CO ₂ (95% air) conditions. The cells were counted and dispensed at 3×10 ² cells/well, and BMP-2 (Bone morphogenetic protein-2) at 100 ng/ml with rutonarin compounds at concentrations of 0.1, 0.3, 1, 3, 10 and 30 uM was treated with the cells and cultured for 72 hours. After completion of culture, in order to compare the formation and differentiation ability of these osteoblasts, Alkaline phosphatase (ALP), a molecular marker related to the differentiation of osteoblasts, was stained and compared (Fig. 1), and the activity of ALP was compared with Thermo Fisher Scientific Alkaline It was measured by a method using Phosphatase Reagents (FIG. 1). At this time, as a negative control (CON), cells treated with DMSO and cultured were used, and as a positive control (POS), cells treated with and cultured with 100 ng/ml BMP-2 were used.

As shown in FIGS. 1 and 2, it was confirmed that the staining level of ALP and the activity of ALP were increased in the experimental group treated with 3, 10 and 30 uM of rutonarin compound compared to the control group. In particular, 30uM of rutonarin compound was treated In one case, it was confirmed that the staining level of ALP and the activity of ALP showed the highest level.

Since it can be determined that the degree of differentiation of osteoblasts is further improved as the staining level of ALP and the activity of ALP increase, it was found that the rutonarin compound of the present invention exhibits a great effect in differentiating osteoblasts to form bone. .

Combining Examples 1 to 2 of the present invention, it can be confirmed that the composition of the present invention can be provided to develop a composition for preventing or treating osteoporosis, since it exhibits osteoblast activity.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without changing its technical spirit or essential features. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not limiting. The scope of the present invention should be construed as including all changes or modifications derived from the meaning and scope of the claims to be described later and equivalent concepts rather than the detailed description above are included in the scope of the present invention.

Claims (6)

A pharmaceutical composition for preventing or treating bone disease, comprising lutonarin or a pharmaceutically acceptable salt thereof as an active ingredient,
The bone disease is selected from the group consisting of osteoporosis, facet bone disease, rickets, osteomalacia, renal osteodystrophy in patients with renal failure, rheumatic bone disease, degenerative bone disease, bone metastatic lesion, and periodontal disease Phosphorus, a pharmaceutical composition.
The pharmaceutical composition according to claim 1, wherein the rutonarin or a pharmaceutically acceptable salt thereof promotes osteoblast differentiation.
Osteoporosis, facet bone disease, rickets, osteomalacia, renal failure, including the step of administering the pharmaceutical composition of claim 1 or 2 to a non-human subject's renal osteodystrophy, rheumatoid bone disease, degenerative bone disease, bone A method for preventing or treating bone metastatic lesions or periodontal disease.
Osteoporosis, facet bone disease, rickets, osteomalacia, renal osteodystrophy in patients with renal failure, rheumatoid bone disease, degenerative bone disease, bone metastasis, containing lutonarin or its food chemically acceptable salt as an active ingredient Health functional food composition for preventing or improving bone metastatic lesion or periodontal disease.
Osteoporosis, facet bone disease, rickets, osteomalacia, renal osteodystrophy in patients with renal failure, rheumatoid bone disease, degenerative bone disease, bone metastasis, containing lutonarin or its food chemically acceptable salt as an active ingredient A feed composition for preventing or improving bone metastatic lesion or periodontal disease.
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