KR20230100822A - Pharmaceutical composition for the prevention or treatment of osteoporosis comprising extract of Osmanthus fragrans - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of osteoporosis, and more particularly, by including an extract of Moksu, inhibiting osteoclast differentiation induced by RANKL, inhibiting inflammatory mediators (nitric oxide), and matrix metealloproteinase ) inhibitory effect, NFκB (nuclear factor kappa B) phosphorylation inhibitory effect, c-fos, NFATc1 and beclin-1 inhibitory effect.

Description

Pharmaceutical composition for the prevention or treatment of osteoporosis comprising an extract of Osmanthus fragrans

The present invention relates to a pharmaceutical composition for preventing or treating osteoporosis and a food composition for preventing or improving osteoporosis.

Bone is a hard tissue in which calcium and phosphorus are calcified, and plays an important role in protecting important organs in the body as well as supporting and protecting the body. In addition, it provides a place for muscles that use strength during physical exercise and a space for bone marrow to produce blood, as well as storing minerals essential for bodily functions.

Bone is a special type of connective tissue composed of calcification of hydroxyapatite and other ions, and is composed of an extracellular matrix composed of Type I collagen, glycoprotein, and proteoglycan. In particular, bone tissue is composed of osteoblasts, osteocytes and osteoclasts. Osteoblasts are bone-synthesizing cells that do not form complete bones, but control the mineralization of osteoids through organic products such as osteoclacin and bone sialoprotein. In addition, osteoblasts synthesize alkaline phosphatase, which is a useful clinical indicator in bone formation activity, along with Type I collagen, which is the main component of bone matrix. On the other hand, osteoclasts are differentiated from monocytes and macrophages and respond to signals for bone resorption. Therefore, during bone development, bone resorption by osteoclasts and formation by osteoblasts form the bone, and this process continues repeatedly throughout life. Bone resorption, especially in a healthy state, is followed by the deposition of new bone, a process called bone remodeling. In addition, bone remodeling releases minerals from the skeleton and removes fine parts of damaged bones. Bone homeostasis is maintained by osteoblasts and osteoclasts.

When bone homeostasis by osteoclasts is imbalanced, bone resorption is excessive, resulting in a disease called osteoporosis, in which bones become less hard and easily breakable even with light impact. While bone strength is determined by bone quality and bone density, it is impossible to measure bone quality, so bone density determines whether or not osteoporosis exists. While bone density is measured at its highest around the age of 30, it decreases by 2% every 5 years thereafter, and after menopause, bone density decreases three times faster. In particular, after menopause, an increase in parathyroid hormone secreted from the parathyroid gland produces a large number of osteoclasts to increase bone resorption, whereas it decreases as extracorporeal secretion of calcium increases. In addition, estrogen functions to inhibit the differentiation of monocytes or macrophages into osteoclasts, and the decrease in estrogen after menopause causes monocytes or macrophages to differentiate into osteoclasts, thereby reducing bone density.

As described above, osteoporosis is a disease closely related to aging, and since it is experienced without any symptoms, it can lead to a fatal problem in which bones are broken.

Until now, in relation to the treatment of osteoporosis, a method of increasing bone density by ingesting calcium, which is a component of bone, has been considered, and it is recommended to supplement vitamin D to increase absorption of calcium in the body. As estrogen decreases in postmenopausal women, estrogen replacement therapy is performed to treat and prevent osteoporosis. In particular, as an active treatment method, treatment substances that strongly inhibit bone resorption, such as bisphosphonate, are used, but in some cases, side effects such as osteonecrosis are accompanied, so taking and treatment are recommended under accurate expert diagnosis. In addition, prevention of osteoporosis by dietary therapy or the like through intake of natural products containing calcium and natural products capable of increasing the absorption of calcium or plant estrogens has been proposed.

On the other hand, woodseo is a woody plant belonging to the plant kingdom, angiosperm, dicotyledon, gentian, and ash, and is weak in temperate southern species, moderate water, and cold resistance, and inhabits in abundance in Korea and China. The flowers of Mokseo are biphasic, blooming in October-November, and are very fragrant. In particular, the flower of the tree is a food ingredient listed on the Food Ingredients List of the Ministry of Food and Drug Safety. Regarding the efficacy of Mokseo, in ancient literature such as the Chinese Medicine Dictionary, Herbal Medicine, Herbal Medicine, etc., it is spicy in taste, warm in nature and non-toxic, so it is used for removing phlegm and removing blood, and for redness, acid value, toothache and bad breath caused by cancer hemorrhoids. It is recorded that

Korean Patent Registration No. 10-1885195

An object of the present invention is to provide a pharmaceutical composition for preventing or treating osteoporosis.

An object of the present invention is to provide a food composition for preventing or improving osteoporosis.

1. Moksu ( Osmanthus fragrans ) A pharmaceutical composition for preventing or treating osteoporosis (osteoporosis) containing an extract.

2. The pharmaceutical composition for the prevention or treatment of osteoporosis according to the above 1, wherein the extract is any one extract selected from the group consisting of flowers, stems, leaves, fruits, and beaks of the order.

3. The pharmaceutical composition for preventing or treating osteoporosis according to the above 1, wherein the extraction solvent of the extract is any one selected from the group consisting of water, alcohol, and mixtures thereof.

4. The pharmaceutical composition for preventing or treating osteoporosis according to 3 above, wherein the alcohol is a C1 to C4 lower alcohol.

5. Woodseo ( Osmanthus fragrans ) A food composition for preventing or improving osteoporosis (osteoporosis) containing an extract.

The composition of the present invention has an osteoclast differentiation inhibitory effect induced by RANKL, an inflammatory mediator (nitric oxide) inhibitory effect, a matrix metealloproteinase inhibitory effect, an NFκB (nuclear factor kappa B) phosphorylation inhibitory effect, c-fos , NFATc1 and beclin-1 inhibitory effect, it can show excellent osteoporosis prevention or treatment effect.

Figure 1 shows the results of the cytotoxicity test of the extract of Mokxeo.
Figure 2a is the result of confirming that the extract of RANKL (Receptor activator of nuclear factor kappa B ligand) inhibits the osteoclastization of bone marrow-derived macrophages, Figure 2b is TRAP osteoclasts differentiated from macrophages of Figure 2a (tartrate resistant acid phos-phatase) was counted after staining and shown in a graph.
Figure 3 is a result confirming that the extract of Mokseo inhibits the osteoclastization of Raw264.7 macrophages by RANKL.
Figure 4 is a result confirming that the extract of Mokseo inhibits the synthesis of nitrogen monoxide (nitric oxide, NO) in Raw264.7 macrophages by RANKL.
Figure 5 is a result confirming that the extract of Mokseo inhibits the activity of matrix degrading enzyme in Raw264.7 macrophages by RANKL.
Figure 6 is a result confirming that the extract of Moxeo inhibits the phosphorylation of NFκB in Raw264.7 macrophages by RANKL.
Figure 7 is a result confirming that the extract of woodseo inhibits the expression of c-fos, NFATc1 and beclin-1 in Raw264.7 macrophages by RANKL.
8 is a result confirming that the extract of Moksu inhibits bone resorption in an osteoporosis animal model.
9 is a result confirming that both the Mokseo leaf extract and the Mokseo flower extract have an effect of inhibiting osteoclastogenesis of bone marrow-derived macrophages.

Hereinafter, the present invention will be described.

The present invention relates to a pharmaceutical composition for preventing or treating osteoporosis.

The pharmaceutical composition includes Osmanthus fragrans extract.

The extract of Myxia may be an extract of a whole plant of Myxia, a part of Myxia, or a material derived therefrom. Specifically, the extract of Myxosa may be any one extract selected from the group consisting of flowers, stems, leaves, fruits and beaks of Myxia, and more specifically, the extract of Myxia may be a flower or leaf extract of Myxus.

The term “extract” refers to a substance obtained by dissolving a substance in a solvent and separating it, and specifically, an extract obtained by the extraction treatment in the above list, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, and a crude product of the extract. It includes extracts of all formulations that can be formed using the extract itself and the extract, such as a purified product, or a mixture thereof.

The extraction solvent of the extract of Mokxeo may be any one selected from the group consisting of water, alcohol, and mixtures thereof. The extraction solvent of the extract of Moxa may be any one selected from the group consisting of water, C1 to C6 alcohols, and mixtures thereof, and the C1 to C6 alcohols are methanol, ethanol, propanol, isopropanol, 1,3-propanediol, butanol. , pentanol, hexanol, and the like. The extraction solvent of the extract may be an alcohol solution, and the alcohol concentration of the alcohol solution is 1 vol% to 99.5 vol%, 10 vol% to 99.5 vol%, 1 vol% to 70 vol%, 1 vol% to 40 vol%, 5 vol% to 25 vol%, 7 vol% to 20 vol%, 5 vol% to 25 vol% or 10 vol% to 20 vol%.

The extraction solvent of the extract of Mokxeo may be a lower alcohol of C1 to C4, and specifically, it may be alcohol.

The extraction method of the extract of Mokxora can be used without limitation as long as it is a method commonly used in the art. The extraction method of the extract may be specifically hot water extraction, cold brew extraction, reflux extraction, filtration or ultrasonic extraction, and more specifically, warmed liquid extraction, pressurized liquid extraction (PLE), ultrasonic assisted extraction (microwave assisted extraction: MAE), subcritical extraction (SE), or a combination thereof. The warm liquid extraction may be reflux extraction. The subcritical extraction may be subcritical water extraction (SWE), and subcritical water extraction is also referred to as superheated water extraction or pressurized hot water extraction (PHWE). .

The extraction temperature of the extract is 4 ° C to 70 ° C, 4 ° C to 50 ° C, 4 ° C to 40 ° C, 4 ° C to 30 ° C, 10 ° C to 70 ° C, 15 ° C to 70 ° C, 20 ° C to 70 ° C, 4 ° C to 50 °C, 10 °C to 50 °C, 4 °C to 40 °C, 4 °C to 30 °C, 10 °C to 40 °C, 10 °C to 35 °C or 10 °C to 30 °C.

The extraction time of the extract of Moxa extract is 1 hour to 2 months, 1 hour to 1 month, 1 hour to 15 days, 1 hour to 10 days, 1 hour to 5 days, 1 hour to 3 days, 1 hour to 2 days, 1 hour to 1 day, 5 hours to 1 month, 5 hours to 15 days, 5 hours to 10 days, 5 hours to 5 days, 5 hours to 3 days, 5 hours to 2 days, 5 hours to 1 day, 10 hours to 1 months, 10 hours to 15 days, 10 hours to 10 days, 10 hours to 5 days, 10 hours to 3 days, or 10 hours to 2 days, and the extraction time may be appropriately selected according to the extraction temperature.

The extraction may be performed by a known method such as filtration to separate the residue and the extract from the tree. The extraction may also include removing the solvent from the obtained extract by a known method such as concentration under reduced pressure. The extraction may also include preparing a dried extract by drying the obtained extract, such as lyophilization.

Osteoporosis is a condition in which bones become thin and weak. Osteoporosis may be caused by the gradual absorption of extracellular matrix constituting bone. Osteoporosis refers to a disease in which bone volume is generally reduced and easily fractures even with light impact, and corresponds to a disease different from degenerative arthritis, which is a joint disease.

Osteoporosis may be caused by bone resorption caused by increased osteoclasts by inducing osteoclastogenesis from macrophages or monocytes by receptor activator of nuclear factor kappa-B ligand (RANKL).

Osteoporosis may be caused by bone resorption caused by the gradual decomposition and loss of extracellular matrix due to the overexpression of matrix degrading enzymes in association with tissue remodeling by osteoclasts.

In one embodiment, the Moksu extract may inhibit osteoclastogenesis of macrophages or monocytes induced by RANKL, an osteoclastization inducer. The osteoclastization may be confirmed by staining the activity of tartrate-resistant acid phosphatase, which is a bone resorption index, as an osteoclast differentiation marker. In addition, the extract may be to inhibit the process of RANKL-induced differentiation of monocytes (macrophages or monocytes, etc.) into osteoclasts having the form of giant cells as multinucleated cells.

In one embodiment, the Moksu extract may inhibit the expression or activity of a matrix degrading enzyme overexpressed by RANKL, an osteoclastization-inducing factor, and the matrix degrading enzyme may be, for example, matrix metalloproteinase or the like.

In one embodiment, the extract may inhibit the expression of inflammatory mediators (eg, nitric oxide, etc.) involved in the differentiation of monocytes or macrophages into osteoclasts. In one embodiment, the extract may inhibit the synthesis of nitric oxide overexpressed by RANKL, an osteoclastization inducer, and inducible nitric oxide synthase, a synthesis inducer thereof.

In one embodiment, the extract may inhibit bone loss when administered to an animal exhibiting symptoms similar to human osteoporosis by ovariectomy. The administration may be oral administration or parenteral administration (eg intravenous, intraperitoneal, subcutaneous, rectal and topical administration).

The composition of the present invention has an osteoclast differentiation inhibitory effect induced by RANKL, an inflammatory mediator (nitric oxide) inhibitory effect, a matrix metealloproteinase inhibitory effect, an NFκB (nuclear factor kappa B) phosphorylation inhibitory effect, c-fos , can exhibit NFATc1 and beclin-1 inhibitory effects. In addition, the composition of the present invention can exhibit a bone resorption inhibitory effect in an osteoporosis animal model. Therefore, in relation to these effects, the composition of the present invention may exhibit osteoporosis preventive or therapeutic effects.

The term “prevention” refers to prophylactic measures that result in any degree of reduction in the likelihood of developing a condition to be prevented or a recurrence or recurrent condition, including minor, substantial or major reduction in the likelihood of developing or recurring the condition, as well as total prevention. Refers to a measure, and the degree of likelihood reduction is at least a minor reduction.

The term "treatment" refers to treatment that results in a beneficial effect on a subject or patient suffering from the condition being treated, including not only cure but also relief of any degree, including minor, substantial, major relief, the degree of relief being At least a slight relief.

The pharmaceutical composition of the present invention 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. , but not limited thereto.

Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, dextrin, maltodextrin, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants, but is not limited thereto.

Solid preparations for oral administration include, but are not limited to, tablets, pills, powders, granules, capsules, etc., such solid preparations may contain at least one excipient such as starch or calcium carbonate in the compound. It is prepared by mixing sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

Liquid preparations for oral use include suspensions, solutions for oral use, 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. . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. 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. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is based on the type, severity, activity of the drug, drug It may be determined according to factors including sensitivity to, time of administration, route of administration and excretion rate, duration of treatment, drugs used concurrently, and other factors well known in the medical field. The pharmaceutical 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 single or multiple times. Considering all of the above factors, it is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by a person skilled in the art.

In the pharmaceutical composition of the present invention, the effective amount may vary depending on the patient's age, sex, and body weight, and is generally 1 to 6000 mg per kg of body weight, preferably 60 to 600 mg may be administered once or divided into three times. there is. However, since it may increase or decrease according to the route of administration, severity of disease, sex, weight, age, etc., the dosage is not limited to the scope of the present invention in any way.

The present invention provides a food composition for preventing or improving osteoporosis.

The food composition contains Osmanthus fragrans extract.

Mokxora extract, Mokxora extract, extraction solvent, extraction method, osteoporosis, and the term "prevention" may be within the above range, but are not limited thereto.

The term “improvement” refers to all actions that improve or benefit the symptoms of suspected or affected individuals.

The food composition may be prepared and processed in the form of tablets, capsules, powders, granules, liquids, pills and the like.

A food composition may contain conventional food additives, and the suitability as a food additive is determined according to the standards and standards for the item in accordance with the General Rules of the Code of Conduct for Food Additives and General Test Methods approved by the Korea Food and Drug Administration, unless otherwise specified. judged by

Items listed in the Food Additives Codex include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, kaoliang pigment, and guar gum; It includes, but is not limited to, mixed preparations such as sodium L-glutamate preparations, alkali additives for noodles, preservative preparations, and tar color preparations.

For example, a food composition in the form of a tablet is obtained by granulating a mixture obtained by mixing the composition with an excipient, a binder, a disintegrant, and other additives in a conventional manner, and then adding a lubricant or the like to compression molding or directly compressing the mixture. can be molded. In addition, the food composition in the form of a tablet may contain a flavoring agent and the like, if necessary.

Among food compositions in the form of capsules, hard capsules can be prepared by filling a mixture obtained by mixing the composition with additives such as excipients in a conventional hard capsule, and soft capsules are a mixture obtained by mixing the composition with additives such as excipients. It can be prepared by filling in a capsule base such as gelatin. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative, and the like, if necessary.

The food composition in the form of a ring may be prepared by molding a mixture obtained by mixing the composition with an excipient, a binder, a disintegrant, etc. by a conventionally known method, and, if necessary, may be coated with sucrose or other coating agent, or starch, The surface can also be coated with a material such as talc.

A food composition in the form of a granule may be prepared by a conventionally known method of a mixture of the composition and an excipient, a binder, a disintegrant, etc., and may contain a flavoring agent, a flavoring agent, and the like, if necessary.

Food compositions include beverages, meat, chocolate, foods, and confectionery. It may be pizza, ramen, other noodles, chewing gum, candy, ice cream, alcoholic beverages, vitamin complexes, and health supplements.

The food composition may be applied orally for use as a nutrient, and the application form is not particularly limited. For example, when administered orally, the daily intake is preferably 5000 mg or less, more preferably 2000 mg or less, and most preferably 500 to 1500 mg or 650 mg daily. When formulated into capsules or tablets, one tablet can be administered with water once a day.

The present invention provides a composition for inhibiting osteoclast differentiation induced by RANKL (Receptor activator of nuclear factor kappa-B ligand).

The composition includes Osmanthus fragrans extract.

Moksu, Moksus extract, extraction solvent, extraction method, extraction temperature and extraction time may be within the above ranges, but are not limited thereto.

The present invention provides a composition for inhibiting nitric oxide (NO).

The composition includes Osmanthus fragrans extract.

Moksu, Moksus extract, extraction solvent, extraction method, extraction temperature and extraction time may be within the above ranges, but are not limited thereto.

The NO may be induced by receptor activator of nuclear factor kappa-B ligand (RANKL).

The composition may be a composition for inhibiting NO in vitro.

The present invention provides a composition for inhibiting matrix metealloproteinase.

The composition includes Osmanthus fragrans extract.

Moksu, Moksus extract, extraction solvent, extraction method, extraction temperature and extraction time may be within the above ranges, but are not limited thereto.

The matrix degrading enzyme may be induced by receptor activator of nuclear factor kappa-B ligand (RANKL).

The composition may be a composition for inhibiting matrix degrading enzyme in vitro.

The present invention provides a composition for inhibiting nuclear factor kappa B (NFκB) phosphorylation.

The composition includes Osmanthus fragrans extract.

Moksu, Moksus extract, extraction solvent, extraction method, extraction temperature and extraction time may be within the above ranges, but are not limited thereto.

The NFκB may be phosphorylated by RANKL (Receptor activator of nuclear factor kappa-B ligand).

The composition may be a composition for inhibiting NFκB phosphorylation in vitro.

The present invention provides at least one composition for inhibition selected from the group consisting of c-fos, NFATc1 and beclin-1.

The composition includes Osmanthus fragrans extract.

Moksu, Moksus extract, extraction solvent, extraction method, extraction temperature and extraction time may be within the above ranges, but are not limited thereto.

At least one selected from the group consisting of c-fos, NFATc1, and beclin-1 may be activated by receptor activator of nuclear factor kappa-B ligand (RANKL).

Hereinafter, examples will be described in detail to explain the present invention in detail.

Example

Preparation Example 1. Preparation of Mokxeo extract 1

After collecting and weighing the leaves, 80% alcohol, an extractable solvent, was added as a food raw material in a volume of 10 times. Thereafter, the supernatant was recovered by first extraction at room temperature for 72 hours, and then second and third extraction was performed in 10 times 80% alcohol at room temperature for one week to recover the supernatant. Thereafter, ethanol was recovered from the recovered supernatant in a distillation apparatus adjusted to 40° C., and then a crude extract of about 1/5 volume of the initial supernatant was recovered. The recovered crude extract was frozen in an ultra-low temperature freezer controlled at -80 ° C, and then freeze-dried in a freeze-drying device to recover a dried extract of about 6% of the weight of the initial tree leaf (6% recovery rate).

Preparation Example 2. Preparation 2 of Moksus extract

An extract was prepared in the same manner as in Preparation Example 1, but using Moksora flowers instead of Moksora leaves.

Experimental Example 1. Cytotoxicity analysis of Moksus extract

MTT assay was performed on mouse fibroblast L929 cells for cytotoxicity and cell viability of the extract of Mokxora. Specifically, after treating the cell line with 0 μg / mL to 200 μg / mL of the extract of Moxeo obtained in Preparation Example 1, the cell line was reacted for 24 hours and performed according to the MTT cytotoxicity assay. 1 is a view showing the results of the cytotoxicity analysis of the extract of the leaves of the neck. As shown in Figure 1, when the L929 cells were administered to the L929 cells by concentration of the leaf extract of the neck, the cytotoxicity did not show a significant difference from the control group to which the leaf extract was not administered.

Experimental Example 2. Inhibition of osteoclastogenesis by Moxus extract upon administration of RANKL, an osteoclast-inducing factor, in bone marrow-derived macrophages of experimental animals

Osteoporosis may occur due to the gradual absorption of extracellular matrix constituting bone as osteoclasts increase in vivo. Such osteoclasts are formed by differentiation into osteoclasts when macrophages or monocytes are exposed to receptor activator of nuclear factor kappa-B ligand (RANKL). Accordingly, in this experimental example, it was confirmed whether or not osteoclastogenesis by the extract was inhibited when RANKL was administered to bone marrow-derived macrophages of experimental animals. Specifically, macrophages were isolated and cultured from the bone marrow of mice, administered with 100 ng/mL RANKL, and simultaneously treated with 25 μg/mL and 50 μg/mL of the extract obtained in Preparation Example 1, cultured for 6 days, and then osteoclasts Differentiated osteoclasts were identified by staining for TRAP (tartrate resistant acid phos-phatase), a differentiation-related biomarker.

As a result, it was confirmed that the RANKL extract inhibited osteoclastogenesis of bone marrow-derived macrophages of experimental animals induced by RANKL (FIG. 2). This suggests that the extract can be usefully used for the prevention and treatment of osteoporosis by inhibiting the differentiation of macrophages exposed to RANKL into osteoclasts.

Experimental Example 3. Inhibition of osteoclastogenesis by Moxus extract when RANKL, an osteoclast-inducing factor, was administered in Raw264.7 macrophages

Raw264.7 cells are known to be differentiated into osteoclasts by RANKL as a mouse macrophage cell line. Accordingly, in this experimental example, it was confirmed whether or not osteoclastogenesis was inhibited by the Mokxus extract when RANKL was administered to Raw264.7 macrophages. Specifically, cultured Raw264,7 macrophages were administered with 100 ng/mL RANKL, treated with 25 and 50 μg/mL extracts at the same time, cultured for 6 days, and then stained with TRAP, a biomarker related to osteoclast differentiation. Differentiated osteoclast cells were identified.

As a result, in the process of differentiation into osteoclasts by administering RANKL, an osteoclast-inducing factor, to Raw264.7 macrophages, it was confirmed that the extract inhibits the differentiation of macrophages derived from the bone marrow of experimental animals into osteoclasts (FIG. 3 ). Specifically, when Raw264.7 macrophages were treated with only RANKL, osteoclasts having multiple nuclei and enlarged can be identified. Display) It can be confirmed that the differentiation process of Raw264.7 cells into osteoclasts is inhibited in a concentration-dependent manner. This suggests that the extract can be usefully used for the prevention and treatment of osteoporosis by inhibiting the differentiation of macrophages exposed to RANKL into osteoclasts.

Experimental Example 4. Anti-inflammatory effect of Mokso extract in Raw264.7 macrophages

In general, osteoclast differentiation is promoted under inflammatory conditions, resulting in increased bone resorption. Therefore, the anti-inflammatory effect can inhibit osteoclastogenesis of monocytes or macrophages to prevent osteoporosis. Therefore, in this experimental example, when RANKL was administered to Raw264.7 macrophages, it was confirmed whether or not the synthesis of nitric oxide, a representative inflammation-inducing factor, was inhibited by the extract of Moxa. Specifically, 100 ng/mL RANKL was administered to cultured Raw264,7 macrophages, and at the same time, the extracts obtained in Preparation Example 1 were treated with 25 μg/mL and 50 μg/mL, and after 24 hours, the culture filtrate was separated to obtain general antibiotics. Griess assay, which is an inflammatory activity assay, was performed to confirm the inhibitory effect of nitric oxide synthesis by the extract of Moksus.

As a result, it was confirmed that the synthesis of the inflammation-inducing factor nitric oxide (NO), which was induced by treating Raw264.7 macrophages with the osteoclast-inducing factor RANKL, was inhibited by the extract in a concentration-dependent manner (FIG. 4) . This indicates that the extract of Mokxora exhibits an anti-inflammatory effect by inhibiting the synthesis of nitric oxide, which is increased in macrophages exposed to RANKL, and inhibits the process of differentiation of macrophages into osteoclasts, which can be usefully used for the prevention and treatment of osteoporosis. suggests

Experimental Example 5. Inhibitory effect of matrix degrading enzyme activity by Mokso extract in RANKL-treated Raw264.7 macrophages

In osteoporosis, the extracellular matrix constituting the bone is gradually degraded and lost due to matrix decomposition, which is expressed from osteoclasts and activated, so that bone resorption proceeds. Therefore, inhibition of the activity of matrix degrading enzyme expressed and activated from osteoclasts inhibits bone resorption, thereby preventing osteoporosis. Therefore, in this experimental example, an experiment was performed to confirm this. Specifically, 100 ng/mL RANKL was administered to the cultured Raw264,7 macrophages, and at the same time, 25 μg/mL and 50 μg/mL of the Moxus extract obtained in Preparation Example 1 were treated and cultured for 6 days. Thereafter, the culture filtrate was recovered and the matrix metealloproteinase activity generated was confirmed through gelatin zymography.

As a result, it was confirmed that Raw264.7 macrophages were treated with RANKL, an osteoclast-inducing factor, to inhibit the activity of matrix degrading enzyme expressed in the process of differentiation into osteoclasts in a concentration-dependent manner by the extract of Mokso (FIG. 5). This suggests that the extract can be usefully used for the prevention and treatment of osteoporosis by inhibiting the activity of matrix degrading enzyme expressed in the process of differentiation of macrophages exposed to RANKL into osteoclasts.

Experimental Example 6. Inhibition of Phosphorylation of NFκB, a Transcription Factor Related to Osteoclastization, by Mokso Extract in Raw264.7 Macrophages Treated with RANKL

RANKL, which induces osteoclastization of macrophages associated with osteoporosis, is known as a ligand for RANK (Receptor activator of nuclear factor kappa B), a specific receptor related to the phosphorylation of NFκB (nuclear factor kappa B), a cell signaling factor. It is known to initiate osteoclastization through NFκB phosphorylation. Therefore, inhibition of NFκB phosphorylation by RANKL in macrophages can prevent osteoporosis by inhibiting osteoclastogenesis. Therefore, in this experimental example, an experiment was performed to confirm this. Specifically, 100 ng/mL RANKL was administered to the cultured Raw264,7 macrophages, and at the same time, 25 μg/mL and 50 μg/mL of the Moxus extract obtained in Preparation Example 1 were treated and cultured for 6 days. Thereafter, it was confirmed whether phosphorylation of NFκB is inhibited by performing western blot using a phosphorylated NFκB-specific antibody after separating the protein by treatment with a cell lysis buffer containing a phosphorylation inhibitor.

As a result, it was confirmed that the phosphorylation of NFκB induced by RANKL, an osteoclast-inducing factor, in Raw264.7 macrophages was inhibited in a concentration-dependent manner by the extract of Moxa (FIG. 6). In addition, it was confirmed through β-actin western blot that the amount of each electrophoresed sample was the same. This suggests that the extract can be usefully used for the prevention and treatment of osteoporosis by inhibiting the process of differentiation of macrophages into osteoclasts by inhibiting the phosphorylation of NFκB by RANKL.

Experimental Example 7. Inhibition of lower biomarkers of NFκB, a transcription factor related to osteoclastogenesis, by Mokso extract in RANKL-treated Raw264.7 macrophages

After phosphorylation of cell signaling factor NFκB by RANKL, which induces osteoclastization of macrophages associated with osteoporosis, c-fos, NFATc1 and beclin-1, which are transcription factors suggested as biomarkers related to osteoclastogenesis, are also expressed. will increase Therefore, it can be suggested that suppression of the expression of phosphorylated NFκB downstream transcription factors by RANKL in macrophages can prevent osteoporosis. Therefore, in this experimental example, an experiment was performed to confirm this. Specifically, 100 ng/mL RANKL was administered to the cultured Raw264,7 macrophages, and at the same time, 25 μg/mL and 50 μg/mL of the Moxus extract obtained in Preparation Example 1 were treated and cultured for 6 days. Thereafter, after treatment with a cell lysis buffer containing a phosphorylation inhibitor to separate proteins, western blotting was performed using c-fos, NFATc1 and beclin-1 specific antibodies.

As a result, it was confirmed that the expression of lower transcription factors of NFκB associated with osteoclastization of macrophages was inhibited (FIG. 7). In addition, it was confirmed through β-actin western blot that the amount of each electrophoresed sample was the same. This suggests that the extract can be usefully used for the prevention and treatment of osteoporosis by inhibiting the process of differentiation of macrophages into osteoclasts by inhibiting the phosphorylation of NFκB by RANKL.

Experimental Example 8. Analysis of osteoporosis preventive effect of Moksus extract in osteoporosis animal models

As osteoporosis progresses, bone density decreases due to gradual bone resorption. Accordingly, the Ministry of Food and Drug Safety Health Functional Food Functional Evaluation Guide "Helpful for Bone/Joint Health" section (Publication registration number 11-1470000-002751-01) suggests an ovariectomized animal model as an osteoporosis animal model. Therefore, in this experimental example, it was analyzed whether the oral administration of the extract of Moxus extract had a preventive or therapeutic effect on osteoporosis in an animal model of ovariectomized osteoporosis.

First, the ovaries of female mice (BALB/c) were resected to prepare an osteoporosis animal model. Thereafter, 5 mg/kg and 10 mg/kg of the extract of Moxus extract obtained in Preparation Example 1 was administered orally to the prepared osteoporosis animal model once a day for 13 weeks. Thereafter, the femurs of the experimental animals were removed, the tissues were fixed in 4% formaldehyde, and radiometric analysis was performed using microCT (small animal tomography device, Quantum GX microCT Imaging System, PerkinElmer).

As shown in FIG. 8, the control group (navie) and the Sham group in which only the abdominal region was incised after ovariectomy without ovariectomy did not undergo bone resorption, whereas ovariectomy was performed osteoporosis animal model (OVX group) confirmed that extreme bone resorption occurred in the trabecular bone inside the femur. However, in the experimental groups in which 5 mg/kg and 10 mg/kg of M. xylem extract were forcibly administered orally, it was confirmed that bone resorption was not achieved and bone density was maintained at the level of the control group when compared to the osteoporosis animal model, respectively.

This suggests that the extract of Mokxeo inhibits bone resorption and has the effect of preventing or inhibiting osteoporosis.

Experimental Example 9. Inhibition of osteoclastogenesis by Mokseo leaf extract or Mokseo flower extract upon administration of RANKL, an osteoclast-inducing factor, in bone marrow-derived macrophages of experimental animals

Through the method described in Experimental Example 2 using the extracts obtained in Preparation Example 1 and Preparation Example 2, both Moxus leaf extract (Fig. 9, Osmanthus leaf) and Moxus flower extract (Fig. 9, Osmanthus flower) are induced by RANKL It was confirmed that it inhibits osteoclastization of bone marrow-derived macrophages of experimental animals (FIG. 9).

Claims (5)

A pharmaceutical composition for preventing or treating osteoporosis, comprising an Osmanthus fragrans extract.
The pharmaceutical composition for the prevention or treatment of osteoporosis according to claim 1, wherein the extract is any one extract selected from the group consisting of flowers, stems, leaves, fruits and beaks of Noxus.
The pharmaceutical composition for preventing or treating osteoporosis according to claim 1, wherein the extraction solvent of the extract is any one selected from the group consisting of water, alcohol, and mixtures thereof.
The pharmaceutical composition for preventing or treating osteoporosis according to claim 3, wherein the alcohol is a C1 to C4 lower alcohol.
A food composition for preventing or improving osteoporosis comprising an Osmanthus fragrans extract.

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