KR20150026517A - The composition of Reynoutria elliptica extract ingredients for treatment and prevention of osteoporosis - Google Patents
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- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/306—Foods, ingredients or supplements having a functional effect on health having an effect on bone mass, e.g. osteoporosis prevention
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Abstract
The present invention relates to a composition for the treatment and prevention of osteoporosis, which contains an extract of Reynouta elliptica, which inhibits osteoclast differentiation and bone resorption which promote bone resorption. The extracts reduce the tartrate-resistant acid phosphatase (TRAP) promoted in osteoclast formation during the differentiation of macrophages (mouse macrophages RAW264.7) into osteoclasts and reduce bone resorption. In addition, carbonic anhydrase is an enzyme that acidifies the resorption lacuna region by secretion of hydrogen ions, and V-ATPase, which is an enzyme that exits the lacuna, allows continuous bone resorption in the resorption lacuna region. . The present invention relates to a composition capable of inhibiting the differentiation of osteoclasts and inhibiting carbonic anhydrase II and V-ATPase expression involved in bone resorption.
Description
The present invention relates to a composition for the treatment and prevention of osteoporosis, which contains an extract of Reynouta elliptica, which inhibits osteoclast differentiation and bone resorption which promote bone resorption. The extracts reduce the tartrate-resistant acid phosphatase (TRAP) promoted in osteoclast formation during the differentiation of macrophages (mouse macrophages RAW264.7) into osteoclasts and reduce bone resorption. In addition, carbonic anhydrase is an enzyme that acidifies the resorption lacuna region by secretion of hydrogen ions, and V-ATPase, which is an enzyme that exits the lacuna, allows continuous bone resorption in the resorption lacuna region. . The present invention relates to a composition capable of inhibiting the differentiation of osteoclasts and inhibiting carbonic anhydrase II and V-ATPase expression involved in bone resorption.
The World Health Organization (WHO) defines osteoporosis as a skeletal disease that increases the risk of fracture by weakening bone strength, and bone strength is determined by bone mass and bone quality. The bone mass is formed in the middle of the twenties or early 30's during the young adult years and the bone loss progresses with age after that. Between the ages of 30s and 50s, the bone mass is usually maintained because the bone resorption of the old bone and the bone formation that forms the new bone are made homeostasis.
Bones are dynamic tissues in which formation and uptake persist, and respond to a variety of stimuli. This bone metabolism involves the destruction and absorption of old bone and the formation of new bone, depending on the degree of activity of osteoblast and osteoclast. In normal cases, bone remodeling is caused by the interaction of these cells . However, such homeostasis of bone resorption and osteogenesis is more likely to result in a sudden bone loss after menopause in women, leading to osteoporosis as the bone loss continues as the osteogenesis function is degraded by aging. Osteoporosis progresses rapidly in the early stages but bone loss occurs due to bone resorption, but osteogenesis is inadequate and it is also determined by the ability of osteoid precursor cells to differentiate into osteoblasts. Therefore, it is necessary to promote differentiation of osteoblast cells or to inhibit osteoclast differentiation to reduce bone resorption.
In recent years, rapid economic growth and medical advances have accelerated the entry into the aging society, and the elderly population suffering from the related chronic diseases is increasing rapidly. Osteoporosis is a typical disease that accompanies an increase in the elderly population. Currently, for the prevention and treatment of osteoporosis, phytoestrogen has been proposed as an alternative for postmenopausal osteoporosis prevention, and other steroid medications are treated in parallel.
Osteoblasts play a role in creating bones, and osteoclasts play a role in bone destruction. The mature osteoclasts are polynuclear cells and originate from hematopoietic stem cells. The differentiation of osteoclasts is carried out by osteoblasts. Osteoprogenitor cells are differentiated by various factors, namely bone morphogenetic protein (BMP) and transforming growth factor (TGF-) Osteoblasts secrete protein and non-protein components and induce minerals to exist as bone cells.
Activation of basic phosphatase, expressed in bone, is required for calcium transport involved in initial calcification in rapidly growing bone with calcified cartilage, and calcium used for calcification is regulated by basic phosphatase (Halech, J., et al., Phil. Trans. Royal Soc. Lond., 302, 91-98, 1983). Osteoblast cells synthesize bone fibers such as collagen or bone fibers such as osteocalcin and calcification occurs when crystals are formed by concentrating phosphoric acid and calcium (Fleish H., et al., Am J Physiol., 200, 1296-1300, 1961).
The osteoblasts produce macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor-kB ligand (RANKL) essential for osteoclast differentiation. Osteoclast precursor cells with one nucleus produce M - stimulated by CSF and RANKL and differentiated into giant polynuclear cells in a continuous differentiation process. The differentiated osteoclasts secrete proteases and the like on the surface of the bone to induce acidification of the bone surface, which breaks down the proteins and minerals, which are constituents of the bone, and destroys the bones.
The most important RANKL that most activates osteoclast is osteoclast formation and activation by binding to RANK, a receptor located in osteoclast precursor cells and osteoclasts. In other words, the precursors of mononuclear cells can be fused to differentiate into multinuclear osteoclasts, which are responsible for bone resorption. Bone resorption is accomplished by dissolving minerals by acidification and dissolving hydrolytic enzymes by dissolving substrate proteins by proteolytic enzymes Collagenase, cysteine protease, and the like are involved in bone resorption (Boyle WJ et al., Nature, 423, 337-342, 2003).
Although the invention of bone resorption inhibitor has been widely used as a preventive and therapeutic agent for osteoporosis, the invention of osteogenesis promoting agent is still insignificant, and the invention of natural bone material for bone resorption inhibitor and osteogenesis promoter is insignificant. In postmenopausal women, It has been reported that effective, but increased incidence of breast cancer and increased atherosclerotic disease, and osteoporosis drugs that require long-term administration are likely to cause side effects.
Although the invention of bone resorption inhibitor has been widely used as a preventive and therapeutic agent for osteoporosis, the invention of osteogenesis promoting agent is still insignificant, and the invention of natural bone material for bone resorption inhibitor and osteogenesis promoter is insufficient. There has been no report on the use of omega-3 fatty acids as a physiologically active substance for treating osteoporosis.
The present inventors have completed the present invention as a therapeutic agent for osteoporosis in which the extract of Lentinula edodes suppresses osteoporosis by inhibiting TRAP activity involved in bone resorption and expression of bone resorption-related enzyme carbonic anhydrase in osteoclast differentiation of macrophages Respectively.
The object of the present invention is to provide a composition for treating and preventing osteoporosis such as osteoporosis by inhibiting osteoclast differentiation involved in bone resorption and reducing the expression of carbonic anhydrase and V-ATPase, .
In order to accomplish the above object, the present invention provides a method of treating osteoporosis and preventing osteoporosis by inhibiting TRAP activity and inhibiting the expression of carbonic anhydrase and V-ATPase in the osteoclast differentiation stage exposed to callus root extract .
According to the present invention, osteoporosis can be treated and prevented by the effect of suppressing osteoporosis by providing a callus root extract.
FIG. 1 shows the results of inhibiting osteoclastogenesis by inhibiting the differentiation of RAW264.7 macrophages into osteoclasts. The figure is the result of TRAP staining to measure osteoclast differentiation.
FIG. 2 is a graph showing absorbance of inhibiting TRAP activity of osteoclasts in the differentiation of RAW264.7 macrophages to osteoclasts.
FIG. 3 is a graph showing the expression of carbonic anhydrase expressed in the bone resorption step of osteoclast by the Western blot method. The blot was quantified by measuring with a densitometer and plotted as a graph. FIG. 4 is a graph showing the expression of V-ATPase in the bone resorption step of osteoclast by the Western blot method. The blot was quantified by measuring with a densitometer and plotted as a graph.
In order to accomplish the above object, the present invention provides a composition for treating and preventing osteoporosis, comprising osteoporosis extract, by inhibiting osteoclast differentiation in macrophages that promote bone resorption.
More specifically,
Hojangun extract inhibits osteoclastogenesis and differentiation in macrophage osteoclast differentiation, inhibits osteoclast TRAP activity, inhibits carbonic anhydrase and V-ATPase expression, and decreases bone resorption. Accordingly, the present invention provides a composition capable of treating and preventing osteoporosis which can reduce bone resorption by inhibiting osteoclast differentiation without side effects.
The Hojokgung extract may be prepared by using a conventional method or may be purchased commercially.
The active ingredient is contained in an amount of 0.001 to 3% based on the total weight of the composition.
Hereinafter, the present invention will be described in more detail.
The present invention relates to a method for inhibiting osteoporosis induced by osteoclast by inhibiting TRAP activity of osteoclasts and inhibiting bone resorption by blocking the expression of carbonic anhydrase and V-ATPase in osteoclast differentiation of macrophages .
The composition for inhibiting the differentiation of osteoclast according to the present invention is not particularly limited in its formulation, but may be specifically beverage, powder, concentrate, capsule, or pharmaceutical formulation. The root extract is used in an amount of 0.001 to 5% by weight based on the total weight of the composition. If the content is less than 0.001% by weight, the effect can not be expected. If the content is more than 5% by weight, safety or formability is difficult. In consideration of effective effects, stability and formulation stability, 0.01 to 3.0% by weight is preferable.
The present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.
(Example 1) Cell culture experiment
(1-1) Culture of mouse macrophages (RAW 264.7)
Mouse macrophages (RAW 264.7) were purchased from ATCC (USA) and cultured. RAW264.7 was prepared by using Dulbecco's Modified Eagles Medium (DMEM, Sigma co., St. Louis, MO, USA) supplemented with 10% FBS, 2 mM glutamine, 100 U / ml penicillin and 100 mg / ml streptomycin 37, and 5% CO 2 . To differentiate into osteoclasts, the extracts were treated with 50 ng / ml RANKL for 5 days.
(Example 2) Inhibition of osteoclast differentiation of T. japonica extract
(2-1) TRAP staining method
For the TRAP staining, 0.110 4 d RAW 264.7 cells were treated with 50 ng / ml RANKL medium. The cultured Triton x-100 was treated for 10 seconds. After removing Triton x-100, the cells were washed with PBS and stained with Leukocyte Acid Phosphatase Assay Kit (Sigma Co.) at 37 for 30 min. Then, osteoclast formation with TRAP was observed using an optical microscope.
The results are shown in Fig.
As shown in FIG. 1, the differentiation of osteoclasts (multi-nucleated cells) was inhibited by treatment of callus root extract in the process of differentiating macrophages with RANKL.
(2-2) Measurement of TRAP activity
For the measurement of TRAP activity, RAW264.7 cells at a density of 0.110 4 and 50 ng / ml RANKL were added to the medium for 5 days. After incubation with 4% formalin solution for 10 minutes, 10 mM citrate buffer (pH 4.6) containing 10 mM sodium tratate and 5 mM p- nitrophenylphosphate was added, incubated at 37 ° C for 1 hour, and then incubated with 0.1 N NaOH at 405 nm absorbance.
The results are shown in Fig.
TRAP activity in TRAP-positive osteoclasts was inhibited by the callus root extract.
(Example 3) Inhibition of the expression of carbonic anhydrase, an enzyme involved in bone resorption,
(3-1) Western blotting of carbonic anhydrase
To measure carbonic anhydrase, which helps bone resorption and acidification of osteoclasts, RANKL and Root Extracts of 50 ng / ml were treated with osteoclast precursor cells (raw 264.7). Western blotting of cell extract proteins was performed by 10% SDS-PAGE. Protein bands obtained by electrophoresis were transferred to nitrocellulose membrane and blocked in 5% skim milk for 3 hours to prevent nonspecific binding. Rabbit anti-rabbit horseradish peroxidase (1: 10000, Jackson ImmunoReasearch Lab.) Secondary antibody was diluted 1,000-fold with primary antibody (rabbit polyclonal antibody, abcam, Lt; / RTI > The carbonic anhydrase protein in the nitrocellulose membrane was detected with Supersignal West pico chemiluminescence (Pierce Biotech, NJ, USA) and photographed with an X-ray film (Konica Co., Tokyo, Japan).
The results are shown in Fig.
RAW 264.7 In the process of differentiation of osteoclast precursor cells, treatment of callus root extract for 5 days together inhibits the expression of carbonic anhydrase in differentiated osteoclasts.
(3-2) Western Blot of V-ATPase
To measure V-ATPase, an enzyme that excretes hydrogen ions from the osteoclasts, RANKL and hyaline root extracts were treated with 50 ng / ml of osteoclast precursor cells (raw 264.7). Western blotting of cell extract proteins was performed by 10% SDS-PAGE. Protein bands obtained by electrophoresis were transferred to nitrocellulose membrane and blocked in 5% skim milk for 3 hours to prevent nonspecific binding. The reaction was carried out with 1: 1000 dilution of goat polyclonal antibody (SANTA CRUZ BIOTECHNOLOGY, USA) of V-ATPase protein and incubated with secondary antibody of rabbit anti-goat horseradish peroxidase (3: 10000, Jackson ImmunoReasearch Lab. Lt; / RTI > The V-ATPase protein on the nitrocellulose membrane was detected by Supersignal West pico chemiluminescence (Pierce Biotech, NJ, USA) and photographed on X-ray film (Konica Co., Tokyo, Japan).
The results are shown in Fig.
RAW 264.7 Treatment of osteoclast precursor cells for 5 days with callus extracts reduces V-ATPase expression in differentiated osteoclasts.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20190092831A (en) | 2018-01-31 | 2019-08-08 | 주식회사 노브메타파마 | Composition for preventing and improving woman climacterium symptoms comprising extract of Polygonum cuspidatum Sieb. et Zucc. and Cinnamomum cassia Blume |
| KR20190107894A (en) | 2018-03-13 | 2019-09-23 | 주식회사 케이오씨바이오텍 | Composition for preventing and improving woman climacterium symptoms comprising Glycine soja seed |
| KR20200038411A (en) * | 2018-10-02 | 2020-04-13 | 고려대학교 산학협력단 | Composition for Preventing or Treating Bone Diseases Comprising IF1 |
| WO2021020857A1 (en) * | 2019-07-30 | 2021-02-04 | 주식회사 노브메타파마 | Use of composition for prevention, alleviation or treatment of bone loss disorders, containing extracts of reynoutria japonica and cassiae cortex interior |
-
2013
- 2013-09-03 KR KR20130105487A patent/KR20150026517A/en not_active Application Discontinuation
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20190092831A (en) | 2018-01-31 | 2019-08-08 | 주식회사 노브메타파마 | Composition for preventing and improving woman climacterium symptoms comprising extract of Polygonum cuspidatum Sieb. et Zucc. and Cinnamomum cassia Blume |
| KR20190107894A (en) | 2018-03-13 | 2019-09-23 | 주식회사 케이오씨바이오텍 | Composition for preventing and improving woman climacterium symptoms comprising Glycine soja seed |
| KR20200038411A (en) * | 2018-10-02 | 2020-04-13 | 고려대학교 산학협력단 | Composition for Preventing or Treating Bone Diseases Comprising IF1 |
| WO2021020857A1 (en) * | 2019-07-30 | 2021-02-04 | 주식회사 노브메타파마 | Use of composition for prevention, alleviation or treatment of bone loss disorders, containing extracts of reynoutria japonica and cassiae cortex interior |
| KR20210014541A (en) * | 2019-07-30 | 2021-02-09 | 주식회사 노브메타파마 | COMPOSITION COMPRISING EXTRACT OF POLYGONUM CUSPIDATUM SIEB. et ZUCC. AND CINNAMOMUM CASSIA BLUME FOR PREVENTING, IMPROVING OR TREATING OF BONE LOSS RELATED DISEASE |
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