KR20190042999A - Composition for Prophylaxis and Treatment of Osteoporosis Comprising Abeliophyllum Distichum Extract - Google Patents

Abstract

The present invention relates to a composition for prevention and treatment of osteoporosis which can be used safely because it inhibits osteoclast differentiation and absorption of glutinous cells and is not cytotoxic, and more particularly relates to a composition for preventing and treating osteoporosis, To a pharmaceutical composition for preventing and treating osteoporosis.

Description

Technical Field [0001] The present invention relates to a composition for preventing and treating osteoporosis,

The present invention relates to a composition for prevention and treatment of osteoporosis, which can be used safely because it inhibits osteoclast differentiation and bone uptake and is not cytotoxic.

Osteoporosis is a skeletal disorder in which the strength of the bones weakens and fractures easily in small impacts. Our body bones are repeatedly absorbed (destroyed) and regenerated throughout their lifes. Bone homeostasis is maintained by balancing bone formation and osteoclast uptake by new osteoblasts . The osteoclasts attach to the surface of the bone and secrete acid and lytic enzymes to dissolve the aged bone by removing the bone matrix that constitutes the bone. On the other hand, osteoblasts secrete calcium and phosphorus to form new bones and maintain the skeleton. These homeostasis of bone resorption and osteogenesis recreates about 10 to 30% of adult bone per year.

Bone homeostasis is regulated by various hormones and other chemical components of the body. When imbalance occurs between osteoblasts and osteoclasts due to hormonal abnormalities and stresses in the body, bone metabolic diseases occur. Bone homeostasis is generally maintained in the 30s to 50s, but postmenopausal women or elderly males induce excessive activity of osteoclasts due to osteoclast differentiation factor, resulting in a loss of balance between bone formation rate and destruction rate Resulting in bone loss and osteoporosis.

Due to the aging of the global population, there is a growing interest in osteoporosis and its treatment, with a market of about $ 130 billion related to the treatment of bone diseases, including osteoporosis. The market for osteoporosis treatments is expected to increase further in the future, and research institutes and pharmaceutical companies around the world are investing heavily in developing bone disease treatments.

Treatment of osteoporosis is traditionally divided into female hormone preparations, bisphosphonate preparations and parathyroid hormones, which are widely used in postmenopausal women. Female hormone preparations are effective in reducing fractures but should be used with caution because they increase the risk of pre-existing and breast cancer. Bisphosphonate preparations are the most widely used treatments, inhibiting the function of osteoclasts and reducing the number of osteoclasts to prevent bone destruction. Parathyroid hormone activates osteoblasts to create new bone, which, unlike other osteoporosis treatments, actually results in an increase in bone tissue. However, it is expensive and there are inconveniences to administer by injection.

Although it can be safely used without side effects, there is much interest in effective preventive and therapeutic agents for osteoporosis. Japanese Patent Registration No. 10-0910622 discloses that Lotus root extract has excellent osteoblastogenesis inhibitory effect owing to osteoclast production and osteoclast inhibition. Japanese Patent Application No. 10-1632111 discloses a compound that inhibits osteoclast formation and bone resorption ability And a composition for treating osteoporosis.

Abeliophyllum distichum is a plant native to Korea and only one species in the world. The name "Mizunetsu" is derived from the fact that the fruit resembles a round fan shape. It was first discovered in Korea in 1919 and then moved to Europe and Japan, and now it is loved as an excellent landscape gardener in many countries. In the United States, varieties that have been modified to improve marine trees have been sold. Although it has not been studied much, it has efficacy with anticancer drug (registered patent No. 10-0706131) or atopy treatment (registered patent No. 10-1729210) because it has limited native areas such as Goesan, Youngdong, Buksan, Is known. Even though it is a rare species, it can be easily breed by breaking, giving up and seeding. Therefore, it is necessary to study the efficacy of the precious tree, which is our precious resource, and to proactively reproduce and utilize it.

Patent No. 10-0910622 Registration No. 10-1632111 Registration No. 10-0706131 Patent No. 10-1729210

It is an object of the present invention to provide a composition which can be safely used without side effects and is effective in preventing or treating osteoporosis or improving symptoms.

In order to accomplish the above objects, the present invention relates to a pharmaceutical composition for preventing and treating osteoporosis, which comprises an extract of Aspergillus as an active ingredient.

The present extracts inhibit the expression of genes or gene markers and proteins involved in osteoclast differentiation in a concentration-dependent manner, and effectively inhibit osteoclast activity and bone resorption in a concentration-dependent manner. On the other hand, it was confirmed that the extracts of Cetaceae extracts did not show cytotoxicity and could be safely used.

Spearmint extract can be prepared by a conventional method for producing plant extracts. That is, the extract can be prepared by extracting and / or fractionating at least one member selected from the group consisting of stem, branch, fruit, root, flower and leaf of watercress tree with water or organic solvent. The stem, branch, fruit, root, flower or leaf may be raw, dried or frozen as it is harvested. Further, it is more preferable to carry out extraction after crushing or crushing for efficient extraction. Any of the methods used in the art for extracting natural products may be used for extraction. For example, cold extraction, heat extraction, ultrasonic extraction, or the like may be used as a method using a juice or a solvent that does not use a separate solvent But is not limited thereto. In the case of extraction using a solvent, it is preferable to use water or a C1 to C4 alcohol or a mixture thereof. In addition, the extract may be further fractionated to further concentrate the active ingredient having efficacy against osteoporosis. In the following examples, the extract was dissolved in water, and then fractionated with petroleum ether and ethyl acetate, followed by fractionation with ethyl acetate. However, the present invention is not limited thereto. The extract obtained by extraction using a juice or a solvent can be used as such, concentrated, or dried. It is a matter of course that conventional drying methods such as spray drying, thermal drying and freeze drying can be used.

The composition of the present invention can be prepared by a method known in the pharmaceutical field for use as a drug for the prophylaxis and treatment of osteoporosis, and can be used as a carrier or a pharmaceutically acceptable carrier, a forming agent, Diluent or the like. The composition of the present invention can be formulated into a preparation for oral or parenteral administration and can be used as a preventive and therapeutic agent for osteoporosis. The dose of the active ingredient according to the present invention can be appropriately selected depending on the degree of absorption of the active ingredient in the body, the form of the preparation, the age, sex and condition of the patient, the degree of symptoms, etc., , And may be administered in divided doses. Typical dosages are 0.001 mg / kg · day to 10 g / kg · day. The composition of the present invention can be safely used without toxicity and side effects, so that it can be safely used for prolonged use even for prophylactic purposes.

In addition, the present invention relates to a health functional food composition for preventing osteoporosis and improving symptoms, which comprises an extract of Helicobida plant as an active ingredient. The above mentioned Heliothis root extract may be added to the health functional food of the present invention in an amount of preferably 0.01 to 100% by weight. The effective dose of the health functional food composition of the present invention can be used in accordance with the effective dose of the pharmaceutical composition, but may be less than the above range for health and hygiene purposes or long-term intake for health control purposes , The active ingredient can be used in an amount in the above range because there is no problem in terms of safety. The health functional food of the present invention includes forms such as tablets, capsules, pills, and liquids. Examples of foods to which the composition of the present invention can be added include various foods, beverages, gums, tea, Alcoholic beverages, chocolate, vitamin complexes, and health functional foods.

As described above, the microalgae extract of the present invention effectively inhibits osteoclast differentiation, osteoclast activity, and bone resorption, but is not cytotoxic, and thus can be effectively used for preventing osteoporosis and improving treatment / symptom.

FIG. 1 is a graph showing an evaluation result of cytotoxicity of Herba extract.
FIG. 2 is a microscope image and graph showing the effect of inhibiting osteoclast differentiation induced by the extracts of Echinochloa crus-galli.
Fig. 3 is an electrophoresis image and graph showing the effect of suppressing the expression of CA2, c-Fos, RANK, and NFATc1, which are genes related to osteoclast differentiation, from the bitter gourd extract.
FIG. 4 is an electrophoresis image and graph showing the effect of inhibiting osteoclast differentiation gene markers CTK, MMP-9,
FIG. 5 is an electrophoresis image and a graph showing the effect of Echinochloa crus-galli extract on the expression of NFATc1 and c-Fos protein in osteoclast differentiation.
FIG. 6 is an electrophoresis image and graph showing the effect of Echinochloa crus-galli extract on the expression of p-ERK, p-JNK, and p-P38 proteins in osteoclast differentiation.
FIG. 7 is a microscopic image and graph showing the effect of the bitterwood extract on osteoclast bone resorption. FIG.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these embodiments are merely examples for explaining the content and scope of the technical idea of the present invention, and thus the technical scope of the present invention is not limited or changed. It will be apparent to those skilled in the art that various changes and modifications can be made within the scope of the technical idea of the present invention based on these examples.

[Example]

Example 1: Preparation of Spruce tree extract

1) Manufacturing process of spearmint extract

To a 500 g dry sample of leaf, branch, flower or immature fruit of the spruce tree, 2 L of 70% (v / v) alcohol was added and extracted with stirring for 3 days. The extract was filtered and the filtrate was concentrated under reduced pressure to about 500 mL. 500 mL of petroleum ether was added to the concentrate and stirred. Then, the upper petroleum ether layer was separated and removed, and 500 mL of petroleum ether was added to the aqueous layer, followed by stirring and separation. 500 mL of ethyl acetate was added to the water layer, and the mixture was stirred. The organic layer was collected three times. The organic layer was recovered and concentrated and dried to obtain an extract.

 Table 1 shows the yields of extracts according to the parts of the trees.

The yields of extracts were different according to the sites. However, since the efficacy against osteoporosis was not large at the time of preliminary examination, leaf extract was used in the following examples.

Example 2: Culturing of cells and differentiation into osteoclasts

1) Cell culture

RAW 264.7 cells were distributed from the Korean Cell Line Bank. RAW 264.7 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin (P / S) at 37 ° C and 5% CO ₂ .

2) Differentiation into osteoclasts

RAW 264.7 cells, a murine macrophage lineage, differentiate into functional osteoclasts when exposed to RANKL (Receptor activator of nuclear factor kappa-B ligand). Thus, RAW 264.7 cells cultured above were treated with RANKL at a concentration of 100 ng / ml to induce osteoclast differentiation. More specifically, cells were seeded with Minimum Essential Medium Alpha (a-MEM) containing 10% FBS and 1% P / S, and then 100 ng / mL of Receptor activator of nuclear factor kappa-B ligand (RANKL) And cultured in one medium for 5 days to induce osteoclast differentiation.

Example 3: Evaluation of cytotoxicity

To measure cytotoxicity, RAW 264.7 cells cultured in 1) of Example 2 were inoculated into a 96-well plate at a concentration of 5 × 10 ³ cells / 100 μL Respectively. The cell viability was measured by MTS assay after treating the cells with 1, 10, 50, and 100 ㎍ / ㎖ of the extracts for 24 hours.

FIG. 1 is a graph showing the results. It shows that the herbaceous plant extract does not show cytotoxicity and can be safely used.

Example 4: Evaluation of efficacy of extracts from Leucocandii for osteoclast differentiation

At the osteoclast differentiation of Example 2, the extracts of Pinus rigicum with RANKL were added at different concentrations and cultured. The cultured cells were assayed for TRAP (Tartrate-resistant acid phosphatase) positive cell number and TRAP activity, which are markers of osteoclast differentiation, to evaluate the efficacy of the extract from the osteoblast differentiation on osteoclast differentiation. After 5 days of incubation, the cells were harvested and fixed with 4% formalin for 10 min. The cells were stained according to the manual of acid phosphatase kit (Sigma aldrich). The TRAP activity of the cell culture medium was determined by adding 50 uL of culture solution to a 92-well plate, adding the same amount of substrate solution (4.93 mg of 4-nitrophenyl phosphate disodium salt hexahydrate, 850 uL of 0.5 M acetate and 150 uL of tartrate) and reacting at 37 ° C for 1 hour. After the reaction, 50 uL of 0.5 M NaOH was added, the reaction was stopped, and the absorbance at 405 nm was measured to show the activity of TRAP as a percentage.

FIG. 2 is a TRAP staining image and a TRAP activity evaluation graph according to the above method. In Fig. 2, the number of TRAP-positive cells was gradually decreased depending on the concentration of Pinus densiflora extract, and the TRAP activity increased by RANKL treatment was also significantly decreased as the treatment concentration of Pinus rigida was increased.

Example 5: Evaluation of the efficacy of the extracts of Alaska pollack on the expression of osteoclast differentiation-related genes

RAW 264.7 cells were cultured for 4 days with RANKL and Sprague-Dawley rats, and the osteoclast differentiation-related genes CAII (carbonic anhydrase) , c-Fos (cellular-fujinami osteosarcoma virus) , RANK The expression of TRAP was measured by rt-PCT in the presence of nuclear factor kappa-B, NFATc1 , cytoplasmic, calcineurin dependent 1, CTK (cathepsin K), MMP-9 (Matrix metallopeptidase 9) Respectively.

For this purpose, RNA was isolated from the cells inducing osteoclast differentiation in Example 2-2) according to the manufacturer's manual in TRIzol (Invitrogen, San Diego, CA, Usa) solution. CDNA was synthesized from isolated RNA using dNTP, oligo dT primer, buffer, dithiothreitol, Superscript II reverse transcriptase and RNase inhibitor. Genes involved in osteoclast differentiation were PCR amplified from the synthesized cDNA using the primers shown in Table 2 below. PCR amplification was repeated 30 cycles at 95 ° C for 30 seconds, at 58-60 ° C for 30 seconds, and at 72 ° C for 30 seconds.

FIG. 3 and FIG. 4 are graphs showing the results. All osteoclast differentiation-related genes were expressed by treatment with RANKL, and the expression was decreased in a concentration-dependent manner upon treatment with the extracts of Herceptin.

Example 6: Evaluation of the efficacy of the extracts of Alaska pollack on the expression of osteoclast differentiation-related proteins

The osteoclast differentiation-related proteins c-Fos, NFATc1, and MAPK (ERK, NFATc1, and ERK) were cultured in RAW 264.7 cells for 5, 15, 30, JNK, P38) was quantitated by Western blotting. Cells incubated for a certain period of time were dissolved in RIPA buffer (50 mM tris-Cl, 150 mM NaCl, 1% NP-40, 0.5% Na.dioxychloate, 0.5% SDS, protease inhibitors and phosphatase inhibitors) 0.0 > rpm, < / RTI > 4 DEG C for 20 minutes. Proteins were quantitated using bicinchoninic acid kit (Sigma-Aldrich). Proteins were separated from 10% SDS-polyacrylamide gel and transferred to nictrocellulose membranes using transfer kit. And the amount of expression was measured.

FIG. 5 shows the expression of c-Fos and NFATc1 proteins after culturing for 24 hours, and FIG. 6 shows the expression of MAPKs according to the elapsed time from 5 minutes to 30 minutes. FIGS. 5 and 6 show electrophoretic images and quantitative graphs showing the results, Lt; RTI ID = 0.0 > protein. ≪ / RTI >

NFATc1 is a protein that regulates osteoclast differentiation by secretion of RANKL, and c-Fos is a protein that mediates RANKL stimulation for osteoclast formation by inducing NAFTc1 as a component of a transcription factor. FIG. 5 shows that the extracts of cetacea extracts inhibit the expression of c-Fos and NFATc1 proteins in a concentration-dependent manner and are effective in controlling osteoclast differentiation.

FIG. 6 shows that MMP-9 inhibits the expression of MMP-9 protein at 5 minutes after treatment with the extract of Echinochloa crus-galli, indicating that it is effective for the transfer of early transcription factors.

Example 7: Evaluation of the efficacy of Bacillus thuringiensis extract on bone resorption ability

RAW 264.7 cells were plated on osteo assay strip well plate and treated with RANKL 100 ng / ㎖ and vinegar extract for 5 days. The pit formed by osteoclasts was analyzed using a phase contrast microscope. 7 is a graph showing a phase contrast microscope image and a pit area as a percentage of the total area in a phase contrast microscope image. Fig. 7 shows that the extracts of the bitter gourd extract inhibit the formation of pit showing bone resorption in a concentration-dependent manner.

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Claims (6)

A pharmaceutical composition for the prevention and treatment of osteoporosis, which comprises Aspergillus oryzae extract as an active ingredient.
The method according to claim 1,
Wherein said extract is selected from the group consisting of stem, branch, fruit, root, flower and leaf.
3. The method of claim 2,
Wherein the stem, branch, fruit, root, flower or leaf is a raw material, dried material or frozen as it is harvested.
4. The method according to any one of claims 1 to 3,
Wherein the extract is extracted with water or a C1 to C4 alcohol or a mixture thereof.
A health functional food composition for preventing and treating symptoms of osteoporosis, which comprises an extract of Alaska pollack as an active ingredient.
6. The method of claim 5,
Wherein said extract is at least one selected from the group consisting of stem, branch, fruit, root, flower and leaf.

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