KR102600931B1 - Pharmaceutical composition for USE IN preventing or treating METABOLIC BONE DISEASE comprising Azulene as an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating bone metabolic diseases containing azulene as an active ingredient. The pharmaceutical composition according to the present invention increases the expression level of osteoblast differentiation marker genes Dlx5 and Runx2, and TNF It has the effect of promoting the differentiation of osteoblasts by inhibiting the inhibitory effect of osteoblast differentiation by -α, so it can be widely used as a medicine or health food for the prevention or treatment of bone metabolic diseases such as osteoporosis.
In addition, it can fundamentally treat osteoporosis by promoting bone formation by increasing the activity of osteoblasts rather than targeting osteoclasts, and has the advantage of showing a secondary fracture prevention effect.

Description

Pharmaceutical composition for preventing or treating bone metabolic diseases comprising Azulene as an active ingredient {Pharmaceutical composition for USE IN preventing or treating METABOLIC BONE DISEASE comprising Azulene as an active ingredient}

The present invention relates to a pharmaceutical composition for the prevention or treatment of bone metabolic diseases containing azulene as an active ingredient, and more specifically, to a pharmaceutical composition for the prevention or treatment of bone metabolic diseases containing azulene as an active ingredient, which promotes osteoblast differentiation. It relates to pharmaceutical compositions.

Recently, various industrial accidents and traffic accidents have increased, and as we enter an aging society, musculoskeletal diseases caused by osteoporosis and subsequent fractures are increasing. To prevent and treat such bone diseases, various types of bone grafting agents are being developed to induce bone regeneration ability, and new types of treatments for related diseases are being developed to improve the activity of osteoblasts and reduce the activity of osteoclasts. In addition, various studies are being attempted using stem cells and osteoblasts to actively induce new bone that can fill areas with bone tumors or severe bone defects.

The bone remodeling process is a process in which old bone is periodically converted into new bone. This process progresses through stages such as proliferation, differentiation, and induction of mineralization of the extracellular matrix. In general, the process of bone formation and resorption in adults maintains bone health by maintaining a balance through the interaction between osteoblasts derived from mesenchymal stem cells and osteoclasts derived from hematopoietic stem cells. However, the activity of osteoblasts decreases and bone formation decreases. If there is an imbalance in activity between osteoblasts and osteoclasts, such as when the activity of osteoclasts becomes stronger and bone resorption increases, there is no significant change in the chemical composition of the tissue, but bone mass decreases, causing bone metabolic diseases such as osteoporosis. You can.

Osteoporosis is a complex disease that is affected by environmental factors such as genetic factors, diet, and lifestyle. In particular, in women, bone loss progresses rapidly due to hormonal changes when they reach menopause.

Osteoporosis treatments currently on the market include vitamin D, female hormones, bisphosphonates, selective estrogen receptor modulators, and calcitonin, but most of them prevent bone loss by reducing the activity of osteoclasts and controlling bone resorption. . However, osteoporosis treatments by inhibiting the activity of osteoclasts are fundamentally not treatments that can treat osteoporosis, so there are limits to a complete cure, and an increase in osteoblast activity that can substantially increase bone density and strengthen bones is essential.

In addition, since osteoporosis cannot be treated with only short-term administration of drugs and is a disease that requires long-term administration of drugs, there is a need for the development of new substances that have excellent efficacy and do not cause side effects even when administered over a long period of time.

Accordingly, research on alternative treatments using active ingredients derived from natural products such as herbal medicines and foods that can promote bone formation while minimizing the side effects of existing treatments and bone loss is being actively conducted.

Meanwhile, Azulene (Bicyclo[5.3.0]decapentaene) can be extracted from chamomile flowers, yarrow, absinthe, etc., and belongs to the non-benzene aromatic compounds. Azulen is an anti-inflammatory drug with few side effects and is used as an oral anti-inflammatory agent and gastric ulcer treatment. It is also used as an ointment because it has the function of soothing the skin, strengthening the skin barrier, protecting the skin, and preventing and treating skin diseases. In addition, it has very low toxicity and is used to treat diseases and allergies in children.

However, to date, no research results related to osteoblast differentiation using azulene and the prevention and treatment of bone metabolic diseases such as osteoporosis have been reported.

The purpose of the present invention is to provide a pharmaceutical composition for the prevention or treatment of bone metabolic diseases containing azulene as an active ingredient at a concentration that increases the activity of osteoblasts and causes fewer side effects even when taken for a long period of time.

Another object of the present invention is to provide a food composition for preventing or improving bone metabolic diseases containing azulene as an active ingredient.

The technical problems to be achieved by the pharmaceutical composition and health functional food according to the technical idea of the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below. There will be.

In order to achieve the above purpose,

The present invention provides a pharmaceutical composition for preventing or treating bone metabolic diseases containing azulene as an active ingredient.

The azulene may be characterized as promoting osteoblast differentiation.

The bone metabolic diseases include bone defects, osteoporosis, osteoporotic fractures, diabetic fractures, nonunion fractures, osteogenesis imperfecta, osteomalacia and fractures resulting therefrom, osteogenesis imperfecta, degenerative bone diseases, malocclusion, osteogenesis imperfecta, bone mass loss, and hip osteoarthritis. It may be characterized as being one or more selected from the group consisting of.

The azulene may be characterized by increasing the expression level of the osteoblast differentiation marker gene Dlx5 (distal-less homeobox 5) or Runx2 (runt-related transcription factor 2).

The pharmaceutical composition may be formulated in the form of powder, granules, tablets, hard capsules, soft capsules, or injections.

Additionally, the present invention provides a food composition for preventing or improving bone metabolic diseases containing azulene as an active ingredient.

The azulene may be characterized as promoting osteoblast differentiation.

The pharmaceutical composition according to the present invention effectively promotes osteoblast differentiation by increasing the expression level of osteoblast differentiation marker genes Dlx5 and Runx2 and inhibiting the inhibitory effect of osteoblast differentiation by TNF-α, thereby preventing bone metabolic diseases such as osteoporosis. It can be widely used as a medicine or health food for the prevention or treatment of.

In addition, it can fundamentally treat osteoporosis by promoting bone formation by increasing the activity of osteoblasts rather than targeting osteoclasts, and has the advantage of showing a secondary fracture prevention effect.

However, the effects that can be achieved by the composition and health functional food according to an embodiment of the present invention are not limited to those mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below. It could be.

In order to more fully understand the drawings cited in this specification, a brief description of each drawing is provided.
Figure 1 shows the results of an MTT experiment confirming the cell survival rate in MC3T3-E1 cells according to the concentration of azulene.
Figure 2 shows the results of confirming the expression of osteoblast differentiation genes in MC3T3-E1 cells cultured for 0, 1, and 2 days after treatment with azulene at a concentration of 25 μM.
Figure 3 shows the results of comparing osteoblast differentiation gene expression in MC3T3-E1 cells treated with azulene, ascorbic acid, and beta-glycerophosphate mixture, respectively, and MC3T3-E1 cells treated with a mixture of them.
Figure 4 shows the results of azulene inhibiting the inhibitory effect of osteoblast differentiation by TNF-α. Osteoblast differentiation is inhibited by TNF-α, but when treated with azulene, osteoblast differentiation increases. You can check that.

Since the present invention can make various changes and have various embodiments, specific embodiments will be illustrated in the drawings and described in detail through detailed description. However, this is not intended to limit the present invention to specific embodiments, and the present invention should be understood to include all changes, equivalents, and substitutes included in the spirit and technical scope of the present invention.

The present invention provides a pharmaceutical composition for preventing or treating bone metabolic diseases containing azulene as an active ingredient.

According to one embodiment, a pharmaceutical composition for preventing or treating bone metabolic diseases containing azulene as an active ingredient may be prepared by including azulene and a pharmaceutically acceptable salt, and may promote osteoblast differentiation and improve bone function. It can be used to improve, treat and prevent bone metabolic diseases.

In the present invention, the term "Azulene" is a type of non-benzenoid aromatic hydrocarbon consisting of cycloheptatriene and cyclopentadiene rings and included in natural products or bioactive substances. It is done.

In the present invention, the term “pharmaceutically acceptable salt” refers to a salt prepared by a conventional method, and can be used without limitation by a method known to those skilled in the art. “Pharmaceutically acceptable salts” include, but are not limited to, salts derived from the following inorganic and organic acids and bases that are pharmacologically or physiologically acceptable: Examples of suitable acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid. , benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, etc. Salts derived from suitable bases may include alkali metals such as sodium, alkaline metals such as magnesium, ammonium, etc.

In the present invention, the term “osteoblast” refers to a cubic cell that forms bone and performs bone formation and remodeling functions. Bone is composed of osteocytes and bone matrix, and osteocytes are further divided into osteoblasts and osteoclasts. Osteoclasts absorb bone matrix by dissolving and decomposing existing bone components for continued bone growth, and osteoblasts form collagen into a lattice shape in the areas decomposed by osteoclasts and add calcium, magnesium, phosphorus, etc. to it. It attaches to create new bone matrix. The composition of the present invention can promote bone formation by promoting differentiation of osteoblasts.

In the present invention, the term "bone metabolic disease" includes without limitation diseases in which bone formation is problematic due to a lack of osteoblasts, such as bone destruction caused by interleukin-1 (IL-1) due to a lack of estrogen, Inflammatory diseases may be included, including osteoporosis caused by excessive bone resorption by osteoclasts, bone metastatic lesions, primary tumors formed in the bone, rheumatoid or degenerative arthritis, and periodontal disease. , a concept that includes diseases that promote bone destruction as pathological bone diseases such as inflammatory alveolar bone resorption disease, inflammatory bone resorption disease, and Paget's disease. Examples of the bone metabolic disease include bone defects, osteoporosis, It may include, but is not limited to, osteoporotic fractures, diabetic fractures, non-union fractures, osteogenesis imperfecta, osteomalacia and fractures resulting therefrom, osteogenesis imperfecta, degenerative bone diseases, malocclusion, osteogenesis imperfecta, decreased bone mass, and hip osteoarthritis. No.

In the present invention, the term "osteoporosis" refers to a condition in which bone strength is weakened due to a decrease in bone quantity and qualitative changes, and the prevention, improvement, and treatment of osteoporosis refers to various diseases caused by decreased bone density and bone loss. It is interpreted to include all.

In the present invention, the term “prevention” refers to all actions that inhibit or delay the onset of the disease by administering a composition containing azulene according to the present invention.

In the present invention, the term “treatment” refers to any action that improves or beneficially changes the symptoms of the disease by administering a composition containing azulene according to the present invention.

In the present invention, the term "administration" means introducing the composition of the present invention into a patient by any appropriate method, and the route of administration of the composition of the present invention is through various oral or parenteral routes as long as it can reach the target tissue. It can be administered in a conventional manner, specifically via oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, intranasal, inhalation, intraocular or intradermal routes. The treatment method of the present invention includes administering the composition of the present invention in a pharmaceutically effective amount. It is obvious to those skilled in the art that the appropriate total daily usage amount can be determined by the treating physician within the scope of sound medical judgment. The specific therapeutically effective amount for a particular patient will depend on the type and degree of response to be achieved, the specific composition, including whether other agents are used as the case may be, the patient's age, weight, general health, gender and diet, and time of administration. It is desirable to apply it differently depending on various factors including the route of administration, secretion rate of the composition, treatment period, drugs used together with or simultaneously with the specific composition, and similar factors well known in the medical field. Therefore, it is desirable to determine the effective amount of the composition suitable for the purpose of the present invention by considering the above-mentioned matters.

In addition, the composition of the present invention is applicable to any animal in need of treatment and prevention of bone metabolic disease, and the animals include not only humans and primates, but also livestock such as cattle, pigs, sheep, horses, dogs, and cats.

The concentration of azulene in the pharmaceutical composition containing azulene of the present invention may be 0.01 μM or more and 25 μM or less. Here, there is a problem of cytotoxicity when the concentration of azulene exceeds 25 μM. Additionally, if the concentration is less than 0.01 μM, there is a problem that the pharmaceutical effect may be minimal.

According to one embodiment, the azulene may be characterized by increasing the expression level of osteoblast differentiation marker genes Dlx5 (distal-less homeobox 5) or Runx2 (runt-related transcription factor 2).

Osteoblast differentiation involves hormones, cytokines such as bone morphogenetic proteins (BMPs), distal-less homeobox 5 (Dlx5), runt-related transcription factor 2 (Runx2), osteocalcin (OC), and alkaline phosphatase (ALP). It is regulated by various transcription factors such as. Among them, Dlx5 and Runx2 are essential genes expressed early in osteoblast differentiation, and the expression of these genes means that osteoblast differentiation is accelerated.

Specifically, Dlx5 (distal-less homeobox 5) is induced by bone and regulates transcription by directly binding to the promoter of Runx2 (runt-related transcription factor 2) and OC (osteocalcin), a late marker gene for osteoblast differentiation. It regulates osteoblast differentiation by regulating gene expression.

Runx2 (runt-related transcription factor 2) is widely known as an important regulator in osteoblast differentiation, and is used in multipotent cells or osteoblast precursor cells, such as ALP (alkaline phosphatase), OC (osteocalcin), and bone sialoprotein. It is a gene that promotes osteoblast differentiation by regulating genes.

The pharmaceutical composition can be administered orally or parenterally during clinical administration, and can be used in the form of a general pharmaceutical preparation.

The pharmaceutical composition may be formulated in the form of powder, granules, tablets, hard capsules, soft capsules, or injections.

More specifically, in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, topical preparations, suppositories, sterile injectable solutions, pre-filled injectable solutions, or lyophilized solutions, respectively, according to conventional methods. It can be formulated in any form, but is not limited thereto.

When formulated, it can be prepared using commonly used diluents or excipients such as 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 the active ingredient plus at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. It can be manufactured by mixing. Additionally, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, etc.

The preferred dosage of the pharmaceutical composition of the present invention can be appropriately selected depending on the patient's condition and weight, severity of symptoms, drug form, administration route and period.

The present invention also provides a method for preventing or treating bone metabolic diseases using azulene. The method for preventing or treating bone metabolic disease includes administering the above-described pharmaceutical composition to a patient.

By administering the above-described pharmaceutical composition to a patient, osteoblast differentiation is activated and bone metabolic diseases such as osteoporosis can be prevented or treated.

The present invention also provides a use of azulene for the prevention or treatment of bone metabolic diseases. The azulene can be used to prevent or treat bone metabolic diseases such as osteoporosis by promoting osteoblast differentiation.

The present invention also provides a food composition for preventing or improving bone metabolic diseases containing azulene as an active ingredient.

In the present invention, the term “food composition” refers to a food that acts favorably on one or more functions of an organism to provide a better state of health, regardless of the nutrients provided to the subject consuming it. As a result, the food composition can be used for the prevention, improvement or treatment of diseases or disease-causing factors.

The food composition of the present invention may be characterized by promoting osteoblast differentiation.

The concentration of azulene in the food composition containing azulene of the present invention may be 0.01 μM or more and 25 μM or less. Here, there is a problem of cytotoxicity when the concentration of azulene exceeds 25 μM. Additionally, if the concentration is less than 0.01 μM, there is a problem that the pharmaceutical effect may be minimal.

In addition to containing azulene as an active ingredient, the food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients like a typical food composition.

Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. The above-described flavoring agents include natural flavoring agents (thaumatin), stevia extracts (e.g. rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.).

In addition, the food composition of the present invention can be preferably formulated as a food composition by including one or more foodologically acceptable or pharmaceutically acceptable carriers in addition to the active ingredients described above.

The formulation form of the food composition may be tablets, capsules, powders, granules, liquids, pills, liquids, syrups, juices, suspensions, emulsions, or drops. For example, for formulation in the form of tablets or capsules, the active ingredient may be combined with an oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, etc. Additionally, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tracacance or sodium oleate, sodium stearate, magnesium stearate, sodium Includes benzoate, sodium acetate, sodium chloride, etc. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, etc. Acceptable pharmaceutical carriers for compositions formulated as liquid solutions include those that are sterile and biocompatible, such as saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and One or more of these ingredients can be mixed and used, and other common additives such as antioxidants, buffers, and bacteriostatic agents can be added as needed. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets.

The food composition of the present invention formulated in the manner described above can be used as a functional food or added to various foods.

Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes, health supplements, etc. There is.

In addition, the food composition contains, in addition to azulene, the active ingredient, various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and thickening agents (cheese, chocolate, etc.), pectic acid, and the like. It may contain salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the food composition of the present invention may contain pulp for the production of natural fruit juice, fruit juice beverages, and vegetable beverages.

Azulene, the active ingredient of the present invention, is a natural substance and has almost no side effects like chemicals, so it can be used safely even when taken for a long period of time for the purpose of providing antioxidant functionality.

In addition, the present invention provides a health functional food for preventing or improving bone metabolism diseases containing azulene as an active ingredient.

The health functional food of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of preventing or improving bone metabolic diseases.

In the present invention, the term "health functional food" refers to food manufactured and processed using raw materials or ingredients with functionality useful to the human body in accordance with the Act on Health Functional Food, and adjusts nutrients or regulates nutrients for the structure and function of the human body. It means ingestion for the purpose of obtaining useful effects for health purposes such as physiological effects.

The health functional food of the present invention may be characterized by promoting osteoblast differentiation.

The concentration of azulene in the health functional food containing azulene of the present invention may be 0.01 μM or more and 25 μM or less. Here, there is a problem of cytotoxicity when the concentration of azulene exceeds 25 μM. Additionally, if the concentration is less than 0.01 μM, there is a problem that the pharmaceutical effect may be minimal.

The health functional food of the present invention may contain common food additives, and its suitability as a food additive is determined in accordance with the general provisions and general test methods of the food additive code approved by the Food and Drug Administration, unless otherwise specified. Judgment is made according to specifications and standards.

Examples of items listed in the Food Additives Code include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as dark pigment, licorice extract, crystalline cellulose, high-quality pigment, and guar gum; Mixed preparations such as sodium L-glutamate preparations, noodle additive alkaline preparations, preservative preparations, and tar coloring preparations are included.

For example, the health functional food in the form of a tablet is made by granulating a mixture of azulene, the active ingredient of the present invention, with excipients, binders, disintegrants and other additives in a conventional manner, and then adding a lubricant and compression molding. Alternatively, the mixture can be directly compression molded. In addition, the health functional food in the form of tablets may contain flavoring agents, etc., if necessary.

Among capsule-type health functional foods, hard capsules can be manufactured by filling a regular hard capsule with a mixture of azulene, the active ingredient of the present invention, with additives such as excipients, and soft capsules can be prepared by mixing the extract with additives such as excipients. It can be manufactured by filling the mixture with a capsule base such as gelatin. The soft capsule may contain plasticizers such as glycerin or sorbitol, colorants, preservatives, etc., if necessary.

The health functional food in the form of a pill can be prepared by molding a mixture of azulene, the active ingredient of the present invention, and excipients, binders, disintegrants, etc., using a known method, and if necessary, coated with white sugar or other coating agents. Alternatively, the surface can be coated with substances such as starch and talc.

Health functional food in granular form can be manufactured into granules by mixing azulene, the active ingredient of the present invention, with excipients, binders, disintegrants, etc., using a known method, and if necessary, flavoring agents, flavoring agents, etc. It may contain.

According to one embodiment, the health functional food may include beverages, meat, chocolate, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes, and health supplements.

According to one embodiment, the health functional food consists of health functional food preparations such as tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies, bars, beverages, gums, and candies. It may be one or more formulations selected from the group.

During the bone formation process, the differentiation of osteoblasts is controlled by the expression of genetic traits and has unique characteristics depending on the culture method. Important signaling systems involved in the differentiation of osteocytes and bone formation are representative transforming growth factor-β (TGF-β), bone morphogenetic protein (BMP), and wint/beta-catenin. (Wnt/β-catenin), fibroblast growth factor (FGF), hedgehog, notch, etc. are known.

This signaling system induces the expression and activation of various transcription factors related to bone formation, such as Dlx5 (Distalless-related homeobox 5) and Runx2 (Runt-related transcription factor 2), and ALP (alkaline phosphatase) during the osteocyte differentiation process. , Id1 (inhibitor of DNA binding 1), and OC (Osteocalcin) are expressed. Ascorbic acid (AA), beta-glycerophosphate (β-GP), dexamethasone, etc. are known as substances that induce differentiation in osteoblasts.

In the present invention, an experiment was conducted to determine the effect of azulene on the proliferation and differentiation of osteoblasts using MC3T3-E1 preosteoblasts, which are similar to osteoblasts present in bone tissue. As a result, azulene was used to determine the effect of azulene on the proliferation and differentiation of osteoblasts. It was confirmed that it had a positive effect on cell proliferation and differentiation.

Hereinafter, the present invention will be described in detail using examples and experimental examples to specifically explain the present invention. However, the examples and experimental examples according to the present invention may be modified into various other forms, and the scope of the present invention is limited. The examples and experimental examples described in detail below will provide a better understanding of the present invention to those with average knowledge in the art. It is provided for complete explanation.

Example

The following experiment was performed to confirm the effect of azulene on the proliferation and differentiation of osteoblasts.

Experimental Example 1: Cytotoxicity test

1-1. Experiment method

(1) Experimental materials and cell culture

To determine whether azulene is cytotoxic to osteoblasts, MTT (3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay was performed.

MC3T3-E1 cells, mouse-derived osteoblasts, were purchased from ATCC CRL-2593 (American Type Culture Collection, Manassas, VA, USA). For maintenance, α-MEM (GibcoBRL, Grandisland, NY, USA) medium was supplemented with 10% FBS (Atlas Biologicals, Fort collins, CO, USA) and 1% antibiotics (100U/mL penicillin-100 μg/mL streptomycin (GibcoBRL)). ) was added and cultured in an incubator at 37°C and 5% CO ₂ conditions.

(2) MTT analysis

The MC3T3-E1 cells were inoculated into a 48 well plate at ⁴ 1Х10 per well, and after culturing for 24 hours, they were treated with azulene at concentrations of 0, 5, 10, 20, 25, 50, 100, and 200 μM for 2 days, 4 days. Each was cultured for one day. MTT (Sigma Aldrich, St. Louis, MO, USA) was treated at a concentration of 0.05 mg/mL and cultured for an additional hour under the same conditions. The medium was then removed, and the insoluble formazan crystals were dissolved in DMSO (Duchefa, Haarlem). , The Netherlands) and the absorbance was measured at 540 nm.

1-2. Experiment result

MC3T3-E1 cells are an osteoblast cell line derived from mice and have metabolic characteristics such as osteoblast proliferation, differentiation, and mineralization, and are used in research related to bone formation. The toxicity of azulene was confirmed in MC3T3-E1 cells through MTT assay.

Figure 1 shows the results of an MTT experiment showing cytotoxicity depending on the concentration of azulene in MC3T3-E1 cells. Referring to Figure 1, as a result of measuring cytotoxicity of azulene at concentrations of 5, 10, 20, 25, 50, 100, and 200 μM, no toxicity was observed at concentrations of 5, 10, 20, 25, and 20 μM, but 50 μM. It was toxic starting from a concentration of .

Experimental Example 2: Expression analysis of osteoblast differentiation markers according to azulene treatment

2-1. Experiment method

(1) RNA extraction and cDNA synthesis

Azulene was treated at a concentration of 25 μM, and the media from each culture dish cultured for 0, 1, and 2 days was suctioned, and then washed with 1 mL of Phosphate Buffer Salin (PBS). Afterwards, cells were lysed by treatment with 300 μL of Trizol, and each sample was recovered.

Each recovered sample was reacted at RT for 5 minutes, then treated with 60 μL of chloroform, reacted at RT for 5 minutes, and then placed in a centrifuge (14,500 rpm, 4°C, 15 minutes).

After centrifugation, 150 μL of the supernatant was transferred to a new tube, 150 μL of isopropyl alcohol was added, inverted, and stored at -20°C.

After centrifugation (14,500 rpm, 4 ℃, 15 minutes), the supernatant was removed, washed with 200 μL of ethanol (70%), centrifuged again (13,000 rpm, 4 ℃, 10 minutes), and the supernatant was removed. It was dried for 5 minutes.

Finally, the pellet (RNA) was dissolved in 20 μL of tertiary distilled water, and RNA was quantified using an Infinite M200PRO (Tecan) device.

Next, the RNA sample quantified using TOPscript RT DryMIX (enzynomics), a cDNA synthesis kit, was added and reacted in a Long Gene device at 50°C for 1 hour and 95°C for 5 minutes, then maintained at 4°C to synthesize cDNA.

(2) Real-time polymerase chain reaction (RT-PCR)

19 μL of a mixture of Forward primer, Revers primer, 2x kit (Emerald), and distilled water (D.W) was added to 1 μL of cDNA, adjusting the total volume to 20 μL.

β-actin, Dlx5, and Runx2 primers (Sigma Aldrich Korea), which are genes that can confirm osteoblast differentiation, were used, and the base sequences of each primer and experimental conditions are shown in Tables 1 and 2.

gene Primer Sequence Forward Reverse β-actin 5'-TTC TTT GCA GCT CCT TCG TTG CCG-3' 5'-TGG ATG GCT ACG TAC ATG GCT GGG-3' Dlx5 5'- CAG AAG AGT CCC AAG CAT CC -3' 5'- GAG CGC TTT GCC ATA AGA AG -3' Runx2 5'- CCG CAC GAC AAC CGC ACC AT -3' 5'- CGC TCC GGC CCA CAA ATC TC -3'

gene reaction conditions Cycle Pre-
Denaturation heat denaturation
(Denaturation) Combination
(Annealing) height
(Extension) Post-
extension hold β-actin 25 cycle 95℃ 5 minutes 95℃ 30 seconds 66℃ 30 seconds 72℃ 30 seconds 72℃ 5 minutes 4℃ Dlx5 30 cycle 95℃ 5 minutes 95℃ 30 seconds 62℃ 30 seconds 72℃ 30 seconds 72℃ 5 minutes 4℃ Runx2 30 cycle 95℃ 5 minutes 95℃ 30 seconds 63℃ 30 seconds 72℃ 30 seconds 72℃ 5 minutes 4℃

2-2. Experiment result

(1) Osteoblast differentiation gene expression results according to azulene treatment time

Azulen confirmed the mRNA levels of marker genes for osteoblast differentiation in MC3T3-E1 cells. MC3T3-E1 cells were treated with 25 μM of azulene and the expression of each gene was confirmed using RT-PCR for each sample cultured for 0, 1, and 2 days.

Figure 2 shows the results showing the expression of osteoblast differentiation marker genes induced by azulene. Referring to Figure 2, it was confirmed that the mRNA expression levels of Dlx5 and Runx2, marker genes for osteoblast differentiation, increased on day 2.

This shows that azulene induces osteoblast differentiation by increasing the expression of marker genes for osteoblast differentiation.

(2) Osteoblast differentiation gene expression results according to condition treatment

Ascorbic acid (AA) and beta-Glycerophosphate (β-GP) are used as substances that induce osteoblast differentiation, and ascorbic acid, beta-glycerophosphate mixture, and azulene are used individually. The gene expression levels of the treated and those treated by mixing them were compared. Here, cells cultured for 2 days at a concentration of 25 μM azulene were used.

Referring to Figure 3, as a result of comparison, Dlx5 and Runx2 appear darker when azulene alone is added and when treated with a mixture of ascorbic acid, beta-glycerophosphate, and azulene, compared to when azulene is not added. could be confirmed.

(3) Confirmation of inhibition of the inhibitory effect of azulene on osteoblast differentiation by TNF-α

To confirm that osteoblast differentiation gene expression is inhibited by TNF-α and that azulene inhibits the inhibition of osteoblast differentiation by TNF-α, ascorbic acid and beta-glycerophosphate mixture alone, ascorbic acid and beta-glycerophosphate The gene expression levels of those treated with the phosphate mixture and TNF-α and those treated with a mixture of both were compared. Here, cells cultured for 2 days at a concentration of 25 μM azulene were used.

Referring to Figure 4, as a result of comparison, the expression of osteoblast differentiation genes Dlx5 and Runx2 was decreased by TNF-α, but when treated together with azulene, both Dlx5 and Runx2 increased. In particular, the expression of Dlx5 increased about two-fold, indicating that azulene inhibits the inhibition of marker gene expression for osteoblast differentiation by TNF-α and induces osteoblast differentiation by increasing gene expression.

As a result, azulene can be used to improve, prevent, or treat bone metabolic diseases such as osteoporosis by promoting osteoblast differentiation.

Meanwhile, azulene according to the present invention can be formulated in various forms depending on the purpose. The following illustrates several formulation methods containing azulene as an active ingredient according to the present invention, but the present invention is not limited thereto.

<Example>

1-1. manufacture of powders

Azulene 500 mg

Lactose 100 mg

Talc 10 mg

The above ingredients are mixed and filled into an airtight bubble to prepare a powder.

1-2. manufacture of tablets

Azulene 500 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above ingredients, tablets are manufactured by compressing them according to a typical tablet manufacturing method.

1-3. Manufacturing of capsules

Azulene 500 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

Capsules are prepared by mixing the above ingredients and filling them into gelatin capsules according to a typical capsule manufacturing method.

1-4. Manufacturing of injectable drugs

Azulene 500 mg

Proper amount of sterile distilled water for injection

Appropriate amount of pH adjuster

It is prepared with the above ingredients per ampoule (2 ml) according to the usual manufacturing method for injections.

1-5. Manufacture of liquid

Azulene 100 mg

10 g isomerized sugar

5 g mannitol

Proper amount of purified water

According to the usual liquid preparation method, add and dissolve each ingredient in purified water, add an appropriate amount of lemon flavor, mix the above ingredients, add purified water, adjust the total to 100 ml by adding purified water, and fill it into a brown bottle. Sterilize to prepare the liquid.

Although the present invention has been described in detail above, the scope of the present invention is not limited thereto, and it is known in the art that various modifications and variations are possible without departing from the technical spirit of the present invention as set forth in the claims. It will be self-evident to those with knowledge.

Claims (7)

A pharmaceutical composition for preventing or treating bone metabolic diseases containing azulene as an active ingredient,
The bone metabolic disease is characterized in that it is one or more selected from the group consisting of bone defects, osteoporosis, osteoporotic fractures, diabetic fractures, nonunion fractures, osteomalacia, osteogenesis disorders, degenerative bone diseases, osteogenesis imperfecta, bone mass reduction, and hip joint reduction. A pharmaceutical composition. According to claim 1,
Azulene is a pharmaceutical composition characterized in that it promotes osteoblast differentiation. delete According to claim 1,
The azulene is a pharmaceutical composition characterized in that it increases the expression level of the osteoblast differentiation marker gene Dlx5 (distal-less homeobox 5) or Runx2 (runt-related transcription factor 2). According to claim 1,
The pharmaceutical composition is characterized in that it is formulated in the form of powder, granules, tablets, hard capsules, soft capsules or injections. A food composition for preventing or improving bone metabolism diseases containing azulene as an active ingredient,
The bone metabolic disease is characterized in that it is one or more selected from the group consisting of bone defects, osteoporosis, osteoporotic fractures, diabetic fractures, nonunion fractures, osteomalacia, osteogenesis disorders, degenerative bone diseases, osteogenesis imperfecta, bone mass reduction, and hip joint reduction. food composition. According to claim 6,
Azulene is a food composition characterized in that it promotes osteoblast differentiation.