KR20220085209A - Composition for Prophylaxis and Treatment of Osteoporosis Comprising Gleditsiae Fructus Extract - Google Patents

Abstract

The present invention relates to the osteoporosis preventive or therapeutic effect and the bone loss and osteoclast differentiation inhibitory effect of the extract of johyeolus, which inhibits osteoclast differentiation and actin ring formation without cytotoxicity, and differentiates transcription of osteoclasts. Since it suppresses the expression of factors, osteoclast differentiation-related genes, bone resorption-related genes, and osteoclast fusion-related genes, it can be usefully used for inhibiting bone loss or for preventing or treating bone diseases.

Description

Composition for the prevention and treatment of osteoporosis comprising an extract of spleen thymus {Composition for Prophylaxis and Treatment of Osteoporosis Comprising Gleditsiae Fructus Extract}

The present invention relates to the effect of preventing or treating osteoporosis and inhibiting bone loss and osteoclast differentiation of an extract of johyeopsis.

Bone remodeling proceeds with the proper activation of osteoclasts that resorb bone and osteoblasts that produce bone. The World Health Organization (WHO) defines osteoporosis as a systemic skeletal disease characterized by reduced bone mass and microstructural abnormalities, resulting in weakened bones and brittle bones. Osteoporosis is a disease that causes a decrease in bone mass per unit volume and an overall decrease in bone mass due to a decrease in the bone matrix, and osteoclasts have the greatest influence on the pathogenesis of osteoporosis. In addition, osteoporosis is a representative geriatric disease, and due to an aging population, osteoporosis is recognized as an important health problem worldwide.

Currently, bisphospnate and selective estrogen receptor modulator (SERM) have been used as treatment methods for osteoporosis. is known In particular, since osteoporosis rarely develops clinical symptoms until fracture occurs, it is difficult to treat it at an appropriate time.

Osteoclasts derived from hematopoietic stem cells are differentiated into multinuclear cells by cytokine called RANKL (Receptor activator of NF-κB ligand), which is necessary for the survival and proliferation of monocytes, which are progenitor cells. In the process of differentiation into mature osteoclasts, tumor necrosis factor receptor-associated factor (TRAF6) is activated through the binding of RANKL and RANK, followed by activation of NFATc1/c-Fos, a transcription factor. In particular, c-Fos induces the expression of NFATc1 as a transcription factor that is increased in the early stage of differentiation. NFATc1 is a very important transcription factor for osteoclast differentiation and promotes the expression of osteoclast-associated receptor (OSCAR), markers of osteoclasts, such as TRAP.

Gleditsiae Fructus (hereinafter referred to as GF) is the fruit of Gleditsia sinensis Lamark or Gleditsia japonica Miquel belonging to Leguminosae. Although it is known to be effective against allergic rhinitis and acute inflammation, it is not known about its therapeutic effect on bone metabolism.

Accordingly, the present inventors confirmed the effect of auxin on osteoclast differentiation-related factors, and as a result, by confirming that auxin extract has an effect of effectively inhibiting the differentiation of osteoclasts, which is known as a major cause of postmenopausal osteoporosis, The present invention was completed by confirming that it can be applied and used to improve various bone diseases including osteoporosis.

Korean Patent Publication No. 10-2019-0117153

It is an object of the present invention to provide a composition for inhibiting bone loss.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating bone disease.

Another object of the present invention is to provide a food composition for preventing or improving bone disease.

Another object of the present invention is to provide a composition for inhibiting osteoclast differentiation.

In order to achieve the above object,

The present invention provides a composition for inhibiting bone loss containing an extract of Gleditsiae Fructus as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of bone disease containing the extract of johyopsis as an active ingredient.

Furthermore, the present invention provides a food composition for preventing or improving bone disease, containing the extract of johyopsis as an active ingredient.

Furthermore, the present invention provides a composition for inhibiting the differentiation of osteoclasts containing the extract of johyopsis as an active ingredient.

The extract according to the present invention, without cytotoxicity, inhibits osteoclast differentiation, inhibits osteoclast actin ring formation, osteoclast differentiation transcription factor, osteoclast differentiation-related gene, bone resorption-related gene, and osteoclast Since the expression of the cell fusion-related gene is suppressed, it can be usefully used for inhibiting bone loss or for preventing or treating bone diseases.

1 is a graph showing the results of confirming the cytotoxicity of the ethanol extract of Johyup (GF).
Figure 2 is a view confirming the osteoclast differentiation ability by the treatment with ethanol extract (GF) johyup.
3 is a view confirming the inhibitory effect of osteoclast actin ring formation by the ethanol extract (GF).
4 is a view confirming the effect of inhibiting protein expression of NFATc1 and c-Fos, which are transcription factors for differentiation of osteoclasts, by treatment with an ethanol extract (GF).
5 is a view confirming the gene expression inhibition effect of osteoclast differentiation-related TRAP and OSCAR, bone resorption-related MMP-9, and osteoclast fusion-related ATP6v0d2 by treatment with ethanol extract (GF).

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the following description, detailed descriptions of well-known techniques known to those skilled in the art may be omitted. In addition, in describing the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description may be omitted. In addition, terms used in this specification are terms used to properly express preferred embodiments of the present invention, which may vary depending on the intention of a user or operator or customs in the field to which the present invention belongs.

Therefore, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated.

The present invention relates to a composition for inhibiting bone loss containing an extract of Gleditsiae Fructus as an active ingredient.

As used herein, the term "extract" refers to a preparation concentrated by squeezing a crude drug with an appropriate leaching solution and evaporating the leachate, but is not limited thereto, but includes an extract obtained by extraction treatment, a diluted or concentrated solution of the extract, It may be a dried product obtained by drying the extract, a crude product thereof, or a purified product. The hyssop extract or Wangbulryuhaeng extract can be prepared using general extraction methods, separation and purification methods known in the art. The extraction method is not limited thereto, but preferably, methods such as hot water extraction, hot water extraction, cold extraction, reflux cooling extraction, or ultrasonic extraction may be used.

In the present invention, the extract can be prepared by fractionation by adding a fractionation solvent to the extract prepared by extraction with an extraction solvent or extraction with an extraction solvent. The extraction solvent is not limited thereto, but water, an organic solvent, or a mixture thereof may be used, and the organic solvent is an alcohol having 1 to 4 carbon atoms, a polar solvent such as ethyl acetate or acetone, hexane or dichloro. A non-polar solvent of methane or a mixed solvent thereof may be used. In addition, preferably water, an alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof may be used, and more preferably a mixed solvent of water and ethanol may be used. In an embodiment of the present invention, extraction was performed using a 30% (v/v) aqueous ethanol solution as the solvent, followed by freeze-drying to prepare a 'cookhyup ethanol extract (GF)'.

In one embodiment, the extract of the present invention is at least one selected from the group consisting of water, anhydrous or hydrous alcohol having 1 to 4 carbon atoms, ethyl acetate, acetone, glycerin, ethylene glycol, propylene glycol and butylene glycol as an extraction solvent. It can be extracted, more preferably extracted with an extraction solvent mixed with water and ethanol.

In one embodiment, the composition of the present invention may inhibit osteoclast differentiation or bone resorption.

In one embodiment, the composition of the present invention may include the Wangbulryuhaeng hot water extract at a concentration of 1 to 1000 μg/ml, preferably 1 to 50 μg/ml, and 5 to 40 μg/ml It may be more preferable to include at a concentration of, but is not limited thereto.

In addition, the present invention relates to a pharmaceutical composition for preventing or treating bone disease containing an extract of Gleditsiae Fructus as an active ingredient.

In one embodiment, the bone disease is selected from the group consisting of osteoporosis, osteopenia, osteolytic metastasis, senile kyphosis, and paget disease. It may be one or more, preferably osteoporosis or osteopenia, and more preferably osteoporosis.

In one embodiment, the composition of the present invention may inhibit the RANK signaling pathway.

In one embodiment, the composition of the present invention can inhibit the expression of a protein or gene associated with osteoclast differentiation. The protein or gene related to osteoclast differentiation may be any one selected from the group consisting of NFATc1 (nuclear factor of activated T cell c1), c-Fos, tartrate-resistant acid phosphatase (TRAP) and OSCAR (osteoclast-associated receptor). have.

In one embodiment, the composition of the present invention can inhibit the expression of genes related to bone resorption. The gene related to bone resorption may be any one selected from the group consisting of matrix methalloproteinase 9 (MMP-9), cathepsin K (CTK), and carbonic anhydrase II (CA2).

In one embodiment, the composition of the present invention can inhibit the expression of genes associated with osteoclast fusion. The osteoclast fusion-related gene may be any one selected from the group consisting of ATP6v0d2 (d2isoform of vacuolar H+-ATPase V0 domain) and DC-STAMP (dendritic cell-specific transmembrane protein).

In one embodiment, the bone disease of the present invention may be caused by hyper-differentiation or hyper-activation of osteoclasts. The differentiation or activation of osteoclasts may be induced by RANKL.

In one embodiment, the bone disease of the present invention may be an increase in activity or expression of a protein or gene related to osteoclast differentiation, and the protein or gene related to osteoclast differentiation is TRAP, NFATc1, c-Fos, It may be one selected from the group consisting of OSCAR and RANK.

In one embodiment, the bone disease of the present invention may be an increase in activity or expression of a protein or gene related to bone resorption, and the protein or gene related to osteoclast differentiation is composed of CTK, MMP-9 and CA2 It may be selected from the group.

In one embodiment, the bone disease of the present invention may be an increase in activity or expression of a protein or gene related to osteoclast fusion, and the protein or gene related to osteoclast differentiation is a group consisting of ATP6v0d2 and DC-STAMP It may be selected from

The pharmaceutical composition of the present invention may further include an adjuvant. The adjuvant may be used without limitation as long as it is known in the art, for example, Freund's complete adjuvant or incomplete adjuvant may be further included to increase the immunity thereof.

As used herein, the term "treatment" means, unless otherwise stated, the disease or condition to which the term applies, or one or more symptoms of the disease or condition, which reverses, ameliorates, inhibits the progression, or It means to prevent, and the term treatment as used herein refers to the act of treating. Accordingly, treatment or therapy for bone disease in a mammal may include one or more of the following:

(1) inhibit the growth of, ie, arrest the development of, bone disease;

(2) preventing the spread of bone disease, ie, preventing metastasis;

(3) alleviating bone disease;

(4) preventing recurrence of bone disease; and

(5) alleviating the symptoms of bone disease (palliating)

As used herein, the term "mammal" refers to a mammal that is the subject of treatment, observation or experimentation, preferably a human.

A composition is indicated to be "pharmaceutically or physiologically acceptable" if the recipient animal can tolerate administration of the composition, or if administration of the composition to that animal is suitable. When the amount administered is physiologically important, the agent can be said to have been administered in a "therapeutically effective amount". An agent is physiologically meaningful if the presence of the agent results in a physiologically detectable change in the recipient patient.

The therapeutically effective amount of the composition of the present invention may vary depending on several factors, for example, the administration method, the target site, the condition of the patient, and the like. Therefore, when used in the human body, the dosage should be determined as an appropriate amount in consideration of both safety and efficiency. It is also possible to estimate the amount used in humans from the effective amount determined through animal experiments. These considerations in determining effective amounts are found, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not to cause side effects, and the effective dose level is determined by the patient's Health status, disease type, severity, drug activity, sensitivity to drug, administration method, administration time, administration route and excretion rate, treatment period, factors including drugs used in combination or concurrently, and other factors well known in the medical field can be determined according to The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

The composition of the present invention may also contain carriers, diluents, excipients or combinations of two or more commonly used in biological agents. A pharmaceutically acceptable carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, for example, Merck Index, 13th ed., Merck & Co. Inc. The compound described in , saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components can be mixed and used, and if necessary, other antioxidants, buffers, bacteriostats, etc. Conventional additives may be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate into injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets. Furthermore, it can be preferably formulated according to each disease or component using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

The pharmaceutical composition of the present invention may be formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, or sterile injection solutions according to conventional methods, respectively. .

As used herein, the term “pharmaceutically acceptable” refers to exhibiting properties that are not toxic to cells or humans exposed to the composition.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additive includes starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, Lactose, mannitol, syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, stearic acid Calcium, sucrose, dextrose, sorbitol and talc and the like can be used. The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight based on the composition, but is not limited thereto.

As used herein, the term "administration" means providing a predetermined substance to a patient by any suitable method, and parenteral administration (eg, intravenous, subcutaneous, intraperitoneal or topical administration according to a desired method) ) or oral administration, and the dosage varies according to the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of disease.

The composition of the present invention may be administered parenterally (eg, intravenously, subcutaneously, intraperitoneally or topically) or orally according to a desired method, and the dosage may vary depending on the age, weight, sex, physical condition, etc. of the subject. is selected taking into account. It is self-evident that the concentration of the active ingredient included in the pharmaceutical composition can be variously selected depending on the subject, and is preferably included in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg/ml. If the concentration is less than 0.01 μg/ml, pharmaceutical activity may not appear, and if it exceeds 5,000 μg/ml, it may be toxic to the human body.

The pharmaceutical composition of the present invention may be formulated in various oral or parenteral dosage forms. Formulations for oral administration include, for example, tablets, pills, hard, soft capsules, solutions, suspensions, emulsifiers, syrups, granules, and the like. crose, mannitol, sorbitol, cellulose and/or glycine), lubricants (eg silica, talc, stearic acid and its magnesium or calcium salts and/or polyethylene glycol). In addition, the tablet may contain a binder such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine, and optionally starch, agar, alginic acid. or a disintegrant such as its sodium salt or a boiling mixture and/or absorbent, coloring, flavoring and sweetening agent. The formulation may be prepared by conventional mixing, granulating or coating methods. In addition, representative formulations for parenteral administration are injection formulations, and water, Ringer's solution, isotonic saline, or suspension can be exemplified as a solvent for the injection formulation. The sterile, fixed oil of the injectable preparation can be used as a solvent or suspending medium, and any non-irritating fixed oil including mono- and di-glycerides can be used for this purpose. In addition, the injection preparation may use a fatty acid such as oleic acid.

In the present invention, the term "prevention" refers to any action that inhibits or delays the occurrence, spread, and recurrence of bone disease or osteoporosis by administration of the pharmaceutical composition according to the present invention.

Furthermore, the present invention relates to a food composition for preventing or improving bone disease containing an extract of Gleditsiae Fructus as an active ingredient.

In one embodiment, the bone disease is selected from the group consisting of osteoporosis, osteopenia, osteolytic metastasis, senile kyphosis, and paget disease. It may be one or more, preferably osteoporosis or osteopenia, and more preferably osteoporosis.

When the composition of the present invention is used as a food composition, the composition may be added as it is or used with other foods or food ingredients, and may be appropriately used according to a conventional method. In addition to the active ingredient, the composition may include a food additive that is pharmaceutically acceptable, and the mixing amount of the active ingredient may be suitably determined according to the purpose of use (prevention, health or therapeutic treatment).

As used in the present invention, the term "food supplement additive" refers to a component that can be supplementally added to food, and is added to manufacture health functional food of each formulation, and those skilled in the art can appropriately select and use it. Examples of food supplement additives include various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents such as natural flavoring agents, coloring agents and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners , pH adjuster, stabilizer, preservative, glycerin, alcohol, carbonation agent used in carbonated beverages, etc., but the above examples are not limited to the type of food supplement additive of the present invention.

The food composition of the present invention may include a health functional food. The term "health functional food" used in the present invention refers to food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. using raw materials or ingredients useful in the human body. Here, 'functionality' refers to obtaining useful effects for health purposes, such as regulating nutrients or physiological effects on the structure and function of the human body. The health functional food of the present invention can be manufactured by a method commonly used in the ordinary technical field, and at the time of the preparation, it can be prepared by adding raw materials and components commonly added in the conventional technical field. In addition, if the formulation of the health functional food is also recognized as a health functional food, it may be manufactured without limitation. The composition for food of the present invention can be prepared in various forms, and unlike general drugs, it has the advantage that there are no side effects that may occur during long-term administration of the drug using food as a raw material, and it is excellent in portability, and the present invention health functional food can be taken as an adjuvant to enhance the effect of arthritis or osteoporosis treatment.

In addition, there is no limitation on the type of health food in which the composition of the present invention can be used. In addition, the composition comprising the extract of the present invention or a fraction thereof as an active ingredient can be prepared by mixing known additives with other suitable auxiliary ingredients that may be contained in health functional foods according to the selection of those skilled in the art. Examples of foods that can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages and There are vitamin complexes and the like, and it can be prepared by adding the extract according to the present invention as a main component to juice, tea, jelly, juice, and the like.

Furthermore, the present invention relates to a composition for inhibiting osteoclast differentiation containing an extract of Gleditsiae Fructus as an active ingredient.

In one embodiment, the osteoclast differentiation may be induced by RANKL.

Since the composition of the present invention is made from natural materials, side effects may be less than that of general synthetic compounds even when used as a pharmaceutical composition or a food composition, so it can be safely included in pharmaceutical compositions and health functional foods to be usefully used.

The present invention will be described in more detail through the following examples. However, the following examples are only intended to embody the contents of the present invention, and the present invention is not limited thereto.

<Example 1> Preparation of extract

Johyup (Gyeongbuk, Korea) purchased through Omni Hub. 1000 ml of 30% (v/v) ethanol aqueous solution was added to 100 g of crude rice, followed by immersion extraction for 3 weeks. The extract was filtered with filter paper (Whatman, No 2). After concentration using a vacuum concentrator, it was freeze-dried and powdered. The finally obtained dried ethanol extract of Gleditsiae Fructus (GF) was 15.9 g, and the yield was 15.9%. The crude ethanol extract (GF) was refrigerated until use.

<Example 2> Confirmation of cytotoxicity

RAW 264.7 cells were cultured in a 100° dish using DMEM medium mixed with 10% FBS alc 1% P/S in an incubator of 95% humidity, 37° C. and 5% CO ₂ environmental conditions. RAW 264.7 cells were seeded in a 96-well plate and cultured for 24 hours. After stabilization, the medium was removed and a fresh medium was added, and the GF powder prepared in Example 1 was filtered through a sterile filter (pore size 0.22 μm) and added to each culture medium at a concentration of 5, 10, 20 or 40 μg/ml. and then incubated for 24 hours. Thereafter, the cell viability was confirmed by measuring the absorbance at 405 nm after treatment with the MTS assay solution. Cytotoxicity was expressed as a percentage relative to the untreated control group.

As a result, the ethanol extract (GF) did not show cytotoxicity at any concentration (FIG. 1).

<Example 3> Confirmation of osteoclast differentiation inhibitory ability

In order to evaluate the osteoclast differentiation inhibitory ability of the ethanol extract (GF of Example 1) according to the present invention, tartrateresistant acid phosphatase (TRAP) staining and TRAP activity were measured.

In order to induce osteoclast differentiation, RAW 264.7 cells were aliquoted into 96-well plates and cultured for 24 hours as in the above example, and then exchanged with a medium supplemented with RANKL (100 ng/ml). GF was added to the medium at a concentration of 5, 10, 20, or 40 μg/ml, and cultured for 5 days while exchanging the same medium every 2 days. Differentiated osteoclasts were stained using a TRAP assay kit. The stained cells were observed using a microscope, and cells with three or more nuclei were counted as multinucleated osteoclasts. Here, cells that were not treated with RANKL were identified as the normal group.

Then, for the measurement of TRAP activity, a reagent prepared by mixing 4.93 mg of 4-Nitrophenyl phosphate disodium salt hexahydrate (sigma aldrich, MO, USA), 850 μL of 0.5 M acetate, and 150 μL of tartrate acid solution, and the culture medium (culture solution) ) 50 μ each was aliquoted into a new 96-well plate, and reacted at 37° C. for 60 minutes. After the reaction was terminated by treatment with 50 μL of 0.5M NaOH, the TRAP activity was confirmed by measuring the absorbance at 405 nm with an ELISA instrument.

As a result, more TRAP-positive cells, ie, multinucleated osteoclasts, were formed in the control group (group treated with RANKL only) compared to the normal group (group untreated with RANKL), but in the experimental group treated with GF, osteoclasts were concentration-dependently It was confirmed that the formation was reduced (Fig. 2). In addition, as a result of measuring TRAP enzyme activity (TRAP activity), the control group showed an increase in TRAP enzyme activity compared to the normal group, while the GF-treated group showed a decrease in TRAP enzyme activity compared to the control group, especially 20 , showed a significant (p<0.05, p<0.01) decrease at a concentration of 40 μg/ml ( FIG. 2 ).

<Example 4> Confirmation of osteoclast actin ring inhibitory effect

In order to evaluate the osteoclast actin ring inhibitory ability of the ethanol extract (GF of Example 1) according to the present invention, immunofluorescence analysis was performed.

As in the above example, RAW 264.7 cells were seeded in a 96-well plate, and after 24 hours, exchanged with a medium containing RANKL (100 ng/ml) and GF 5, 10, 20 or 40 μg/ml and cultured for 5 days. did Thereafter, the same medium was exchanged every 2 days and cultured until mature osteoclasts were formed and stabilized. Thereafter, the medium was removed, fixed with 4% formaldehyde, and treated with 0.1% Triton X-100 (in PBS) for 1 minute to increase cell permeability. Then, in order to visualize the actin rings, the F-actin rings were stained with Acti-stain™ 488 Fluorescent Phalloidin, and then observed using an immunofluorescence microscope.

As a result, it was found that the control group (RANKL-treated group) formed a mature actin ring compared to the normal group (RANKL untreated group), whereas the GF-treated experimental group suppressed the actin ring formation (FIG. 3).

<Example 5> Confirmation of the inhibitory effect of osteoclast differentiation transcription factor expression

The effect of the ethanol extract (GF of Example 1) according to the present invention on the expression of a key mechanism protein for osteoclast differentiation was confirmed by the following method.

As in the above example, RAW 264.7 cells were seeded in a 96-well plate, and after 24 hours, RANKL (100 ng/ml) and GF 40 μg/ml were exchanged with a medium and cultured for 1 day. Then, in the cultured cells, protease inhibitor and phosphatase inhibitor RIPA buffer (50 mM Tris.Cl, 150 mM NaCl, 1% NP-40, 0.5% Na. deoxycholate and 0.1% SDS) were used to extract proteins from the cells.

The extracted protein was quantified by BCA protein assay, and then electrophoresed on a 10% polyacrylamide/SDS gel to detect primary and secondary antibody reactions through western blot assay, and then transferred to a nitrocellulose transfer membrane. After that, the membrane was blocked with 5% skim milk dissolved in TBST (0.05% Tween-20 in Tris-buffered saline, pH 7.4) and reacted with anti-NFATc1 antibody and anti-c-Fos antibody, followed by reaction with a secondary antibody. made it Then, it was developed with an ECL solution (Santacruz, California USA) and developed on an X-ray film to confirm the protein expression level. Protein expression levels of NFATc1 and c-Fos were quantified through Actin.

As a result, the control group treated with RANKL increased the expression of NFATc1 and c-Fos proteins compared to the normal group, and it was confirmed that the expression of NFATc1 and c-Fos in the experimental group treated with GF was significantly decreased compared to the control group ( Fig. 4). When this was quantified through Actin, the expression of NFATc1/c-Fos in the GF-treated experimental group was significantly inhibited at a concentration of 40 μg/ml (p<0.05, p<0.01).

<Example 6> Confirmation of expression of genes related to osteoclast differentiation and bone resorption

The effect of the ethanol extract (GF of Example 1) according to the present invention on the expression of genes (mRNA) related to the core mechanism of osteoclasts was confirmed by the following method.

As in the above example, RAW 264.7 cells were seeded in a 96-well plate, and after 24 hours, exchanged with a medium containing RANKL (100 ng/ml) and GF 5, 10, 20 or 40 μg/ml and cultured for 4 days. did Total RNA was extracted by treating the cultured cells with Trizol solution (Invitrogen, USA). After the extracted RNA was quantified at 2 μg through a nanodrop device, it was synthesized as cDNA (complementary DNA) using reverse transcriptase. The synthesized cDNA was amplified by PCR using Taq polymerase, and real-time PCR was performed using the primers in Table 1 below. Thereafter, the resultant was electrophoresed on a 1.2% agarose gel and stained with SYBR Green. The expression level of each gene was normalized to the mRNA concentration using GAPDH (Glyceraldehyde 3-phosphate dehydrogenase), and the density of each band was analyzed using Image J software (Ver. 1.46, National Institutes of Health). .

Primer name Sequence TRAP Forward 5'-ACT TCC CCA GCC CTT ACT ACC G-3' Reverse 5'-TCA GCA CAT AGC CCA CAC CG-3' OSCAR Forward 5'-CTG CTG GTA ACG GAT CAG CTC CCC AGA-'3 Reverse 5'-CCA AGG AGC CAG AAC CTT CGA AAC T-'3 MMP-9 Forward 5'-CGA CTT TTG TGG TCT TCC CC-'3 Reverse 5'-TGA AGG TTT GGA ATC GAC CC-'3 ATP6v0d2 Forward 5'-ATG GGG CCT TGC AAA AGA AAT CTG-'3 Reverse 5’-CGA CAG CGT CAA ACA AAG GCT TGT A-‘3 GAPDH Forward 5’-ACT TTG TCA AGC TCA TTT CC-‘3 Reverse 5’-TGC AGC GAA CTT TAT TGA TG-‘3

As a result of analyzing the effect of GF on the mRNA expression of TRAP and OSCAR, which are factors related to osteoclast differentiation, MMP-9, which is an indicator of a bone resorption-related mechanism, and ATP6v0d2, which is an indicator of a mechanism related to osteoclast fusion, in the control group, TRAP, MMP- 9, mRNA expression of ATP6v0d2 and OSCAR increased significantly compared to the normal group, whereas the expression of TRAP, MMP-9, ATP6v0d2 and OSCAR was inhibited in a concentration-dependent manner in the GF-treated group, especially at 20 and 40 μg/mL concentrations. It was found that expression was significantly suppressed (p<0.05, p<0.01) (Fig. 5).

Claims (16)

A composition for inhibiting bone loss containing an extract of Gleditsiae Fructus as an active ingredient. According to claim 1, wherein the extract is water, anhydrous or hydrous alcohol having 1 to 4 carbon atoms, ethyl acetate, acetone, glycerin, ethylene glycol, propylene glycol, and at least one extraction solvent selected from the group consisting of butylene glycol extracted with , A composition for inhibiting bone loss. The composition for inhibiting bone loss according to claim 1, which inhibits osteoclast differentiation or bone resorption. A pharmaceutical composition for preventing or treating bone diseases, comprising an extract of Gleditsiae Fructus as an active ingredient. 5. The method of claim 4, wherein the bone disease is selected from the group consisting of osteoporosis, osteopenia, osteolytic metastasis, senile kyphosis, and paget disease. One or more, a pharmaceutical composition for preventing or treating bone disease. The pharmaceutical composition for preventing or treating bone disease according to claim 4, which inhibits the RANK signaling pathway. The pharmaceutical composition for preventing or treating bone disease according to claim 4, which inhibits the expression of a protein or gene related to osteoclast differentiation. The method according to claim 7, wherein the protein or gene related to osteoclast differentiation is selected from the group consisting of NFATc1 (nuclear factor of activated T cell c1), c-Fos, tartrate-resistant acid phosphatase (TRAP), and osteoclast-associated receptor (OSCAR). Any one selected, a pharmaceutical composition for preventing or treating bone disease. The pharmaceutical composition for preventing or treating bone disease according to claim 4, which suppresses the expression of genes related to bone resorption. The pharmaceutical composition for preventing or treating bone disease according to claim 9, wherein the gene related to bone resorption is any one selected from the group consisting of MMP-9 (matrix methalloproteinase 9), CTK (Cathepsin K) and CA2 (carbonic anhydrase Ⅱ). . The pharmaceutical composition for preventing or treating bone disease according to claim 4, which inhibits the expression of genes related to osteoclast fusion. The method of claim 11, wherein the osteoclast fusion-related gene is any one selected from the group consisting of ATP6v0d2 (d2isoform of vacuolar H+-ATPase V0 domain) and DC-STAMP (dendritic cell-specific transmembrane protein), bone disease prevention or treatment pharmaceutical composition for use. The pharmaceutical composition for preventing or treating bone disease according to claim 4, wherein the bone disease is caused by osteoclast hyperdifferentiation or hyperactivation. A food composition for preventing or improving bone disease containing an extract of Gleditsiae Fructus as an active ingredient. 15. The method of claim 14, wherein the bone disease is selected from the group consisting of osteoporosis, osteopenia, osteolytic metastasis, senile kyphosis, and paget disease. One or more, a food composition for preventing or treating bone disease. A composition for inhibiting osteoclast differentiation containing an extract of Gleditsiae Fructus as an active ingredient.

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