KR20160141528A - Composition for the prevention and treatment of osteoporosis containing white Hibiscus syriacus L. flower extract - Google Patents

Composition for the prevention and treatment of osteoporosis containing white Hibiscus syriacus L. flower extract Download PDF

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KR20160141528A
KR20160141528A KR1020150077339A KR20150077339A KR20160141528A KR 20160141528 A KR20160141528 A KR 20160141528A KR 1020150077339 A KR1020150077339 A KR 1020150077339A KR 20150077339 A KR20150077339 A KR 20150077339A KR 20160141528 A KR20160141528 A KR 20160141528A
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osteoporosis
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김영옥
박춘근
이상원
김형돈
안영섭
김진성
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대한민국(농촌진흥청장)
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Abstract

The present invention relates to a composition for the prevention and treatment of osteoporosis, which comprises an extract of white mugwort flowers as an active ingredient. The composition is effective for inhibiting osteoclast differentiation, increasing bone density and bone content, The present invention also relates to a composition for preventing and treating osteoporosis which can be widely used as a medicament and a health food useful for prevention or treatment of osteoporosis. INDUSTRIAL APPLICABILITY According to the present invention, there is exhibited a combined effect of simultaneously increasing the effect of increasing the bone density and the bone content and densifying the microstructure of the bone. Particularly, when the concentration of the extract of Mugung flower is 30 ~ 150 ㎍ / ㎖, the effect of inhibiting the differentiation activity of osteoclast is remarkably suppressed without showing cytotoxicity. Thus, it is widely used as medicine or health food useful for prevention and treatment of osteoporosis .

Description

TECHNICAL FIELD The present invention relates to a composition for prevention and treatment of osteoporosis, which comprises an extract of white mugwort flowers as an active ingredient, and a composition for prevention and treatment of osteoporosis comprising white Hibiscus syriacus L. flower extract.

The present invention relates to a composition for the prevention and treatment of osteoporosis, which comprises an extract of white mugwort flowers as an active ingredient. The composition is effective for inhibiting osteoclast differentiation, increasing bone density and bone content, The present invention also relates to a composition for preventing and treating osteoporosis which can be widely used as a medicament and a health food useful for prevention or treatment of osteoporosis.

A bone is a calcified connective tissue associated with muscles. The surface is a thick, hard calcified tissue that plays a role in balancing the body and protects organs. The inside of bone is bone marrow tissue, which is the center of calcium metabolism. The bone consists of an organic substance such as collagen, osteocalcin, osteonectin, and inorganic substance such as calcium, phosphorus, and fluorine, and moisture. In the living body, bone formation and bone resorption are always occurring. Bone formation is promoted by small amounts of parathyroid hormone, androgens, estrogens, fluorine, phosphorus, and bone resorption is promoted by physical decompression, weightlessness, a large amount of parathyroid hormone, corticosteroids, Therefore, balance between them is important in bone metabolism.

Osteoporosis is a disease caused by the collapse of the balance between bone resorption and bone formation, which is caused by excessive bone resorption over osteogenesis. Osteoporosis is a disease caused by reduced lime of bone tissue and thinness of bone, As the bone marrow cavity is widened and the symptoms progress, the bone is weakened, so it is likely to fracture even in small impacts. Bone tissue is a dynamic tissue that is formed by osteoblasts and is constantly repetitively absorbed and destroyed by osteoclasts.

The homeostasis of bone is maintained by continuously controlling the bone remodeling by the equivalent action of bone resorption by osteoclast and osteoblast formation (bone formation) .

However, excessive activity of osteoclasts or a decrease in osteoblast activity leads to an imbalance in the remodeling process, leading to adult skeletal diseases such as osteoporosis. In order to alleviate this imbalance, methods for suppressing excessive activity of osteoclasts, promoting osteoclast activity, inhibiting osteoclast activity and promoting osteoclast activity are generally used, and they are used for treatment of osteoporosis As well as for the treatment of the disease.

Osteoclasts are cells derived from mononuclear cells of hematopoietic stem cells. Mouse RAW264.7 mononuclear cells are differentiated into multinucleated osteoclasts by fusing with RANKL (receptor activator of nuclear factor κB (RANK) ligand) : Boyle WJ, Simonet WS, Lacey DL, Osteoclast differentiation and activation, Nature, 2003 May 15; 423 (6937): 337-42). This differentiation process promotes the activation of mitogen-activated protein kinase (MAPK) by RANKL binding to RANK in the extracellular region, and this is because the transcription factor NF-κB enters into the nucleus and binds to osteoclast differentiation-related TRAP tartrate-resistant acid phosphatase,

MMP-9 (matrix metalloproteinase-9), and c-Src tyrosine kinase. The polynuclear osteoclast formed by this process can absorb mineralized bone. Furthermore, binding of RANKL to RANK promotes the activity of TRAF6 (tumor necrosis factor receptor-associated factor 6)

MAPK, or NF-κB, AP-1, and NFATc1 (Lee ZH, Kim HH, Signal transduction by receptor activator of nuclear factor kappa B in osteoclasts. Biochem Biophys Res Commun. 2003 May 30; 305 (2): 211-4). In particular, it has been reported that AP-1 or NFAT, which is specific for osteoclast differentiation, can be regulated by ERK and NF-κB which are found to be important signaling molecules in osteoclast survival and bone resorption ability. Thus, blockade of the signaling pathway activated by RANKL has been recognized as one of the therapeutic approaches for the treatment of bone diseases including osteoporosis.

Osteoporosis is a cause of 15% of the deaths of the elderly as a result of various fractures, especially femoral fractures or vertebral fractures, which are easily caused by the weakening of the bone rather than the symptom itself, . The bone mass is affected by various factors such as genetic factors, nutritional intake, hormonal changes, exercise and lifestyle differences, and the causes of osteoporosis include age, lack of exercise, low birth weight, smoking, low calcium diet, Ovariectomy and the like are known.

Although there are individual differences, blacks have a lower bone resorption level than blacks, and bone mass is higher. Usually, bone mass is highest at 14-18 years old and decreases about 1% per year at old age. Especially in women, bone reduction continues after 30 years of age, and bone turnover is rapidly progressed by hormonal changes in menopause.

Thus, osteoporosis is an unavoidable symptom for elderly people, especially postmenopausal women. In the developed countries, as population ages, interest in osteoporosis and its therapeutic agents is gradually increasing.

According to the World Health Organization (WHO), osteoporosis is a systemic skeletal disorder characterized by decreased bone mass and microstructural abnormality, lowering bone mass and bone density, and increasing the risk of fracture. In addition, the bone is replaced by a 10% change every year in organs that change over the course of a lifetime, and all bones in the body are replaced with new bones in 10 years. Bone mineral density is highest in the 20s to 30s, and gradually decreases after that, and women become abruptly weak during the first 5 years of menopause.

The osteoclast from the hematopoietic stem cells results in the destruction or resorption of old bone, which is released into the bloodstream and used to maintain body function. On the other hand, the osteoblast produced from the mesenchymal stem cells undergoes new bone formation to rebuild the skeleton. The balance of these bone cells is regulated by a hormone or chemical component mixture. Parathyroid hormone and vitamin D stimulate bone resorption, while estrogen and calcitonin stimulate osteoblasts to form new bone. These osteoporotic osteoarthritis and osteochondral osteoarthritis maintain the bone homeostasis. The faster the osteoarthritis speeds, the weaker the bone becomes, and the fracture becomes easy.

The important cytokine that activates osteoclasts is the Receptor activator of nuclear factor-κB ligand (RANKL), which is produced in osteoblasts or in activated immune cells. The resulting RANKL binds to a receptor (RANK) located in osteoclast and progenitor cells to promote osteoclast formation and activity. There is also an in vivo antagonist, Osteoprotegerin (OPG), for RANKL. Finally, osteoclast formation and activity are regulated by the balance of RANKL and OPG. However, when the cause of stimulation of bone resorption (estrogen deficiency, hyperparathyroidism, etc.) occurs, balance of OPG and RANKL is broken and osteoclast is activated and bone resorption is promoted. Activated osteoclasts attach to the bone surface and secrete strong osteolytic substances. One of the bone protein solubilizers, cathepsin K, is produced and secreted specifically by osteoclasts. There are abundant calcitonin receptors (Cal-R) and TRAP. , and an activating ring to absorb bone matrix. Therefore, the inhibition of bone resorption may be achieved through their inhibition. In order to inhibit bone resorption, studies are under way to inhibit the bone resorption process by inducing early stage inhibition of differentiation blocking osteoclast differentiation from osteoclast precursor cells and differentiating osteoclast cells. Calcium, vitamin D, calcitonin, female hormone, tibolone, selective estrogen receptor modulator (SERM), bisphosphonate and the like have been used as inhibitors of bone resorption. However, these therapies have been reported to have some adverse effects such as atypical fractures, weight gain, gastrointestinal disorders, endometrial cancer and breast cancer, and mandibular necrosis.

In recent years, studies on the relationship between osteoporosis and oxidative stress have been conducted, showing a significant negative correlation between increased oxidative stress and bone mineral density. In women with osteoporosis, serum antioxidant levels were reduced and bone mineral density was increased by antioxidant vitamin intake. Therefore, it is urgently required to prevent or treat osteoporosis by increasing bone density by minimizing bone loss using safe natural materials of effective medicinal plants having antioxidant components.

It is an object of the present invention to provide a method for the treatment of osteoporosis and osteoporosis which comprises a white mugwort flower extract as an active ingredient and has an effect of inhibiting the activity of osteoclasts, an effect of increasing bone density and bone content, A pharmaceutical composition for preventing or treating osteoporosis, or a health functional food for preventing and improving osteoporosis.

Accordingly, the present invention provides a pharmaceutical composition for prevention and treatment of osteoporosis, which comprises a white mugwort flower extract as an active ingredient.

In a preferred embodiment of the present invention, the concentration of the white mugwort flower extract may be 30 to 150 ppm.

In a preferred embodiment of the present invention, the concentration range is a concentration of the stability level that does not affect the survival rate of the osteoclast treated with the composition, and the TRAP activity of the osteoclast may be decreased in the concentration range.

In a preferred embodiment of the present invention, the composition may reduce expression of at least one selected from the group consisting of Cal-R mRNA and TRAP mRNA.

Another aspect of the present invention provides a health functional food for osteoporosis prevention and improvement comprising the pharmaceutical composition for preventing and treating osteoporosis.

Yet another aspect of the present invention is a method for the preparation of a medicinal herb comprising the steps of: washing white Mugwort flowers, drying and crushing them; Adding the solvent to the pulverized white mugwort flower at a volume ratio of 2 to 4; And extracting and cooling the extract, and concentrating the extract to prepare a white mugwort flower extract. The present invention also provides a method for preparing white mugwort flower extract for the prevention and treatment of osteoporosis.

In a preferred embodiment of the present invention, the solvent may include at least one selected from the group consisting of water and C 1 -C 4 alcohols.

In one preferred embodiment of the present invention, the extraction in step 2 may be repeated at 80 ° C to 100 ° C for 1 to 5 hours at least twice.

In one preferred embodiment of the present invention, the concentration in step 3 may be performed at 40 ° C to 50 ° C.

In one preferred embodiment of the present invention, the yield of the white mugwort flower extract may be 30% to 35%.

INDUSTRIAL APPLICABILITY According to the present invention, there is exhibited a combined effect of simultaneously increasing the effect of increasing the bone density and the bone content and densifying the microstructure of the bone. Particularly, when the concentration of the extract of Mugung flower is 30 ~ 150 ㎍ / ㎖, the effect of inhibiting the differentiation activity of osteoclast is remarkably suppressed without showing cytotoxicity. Thus, it is widely used as medicine or health food useful for prevention and treatment of osteoporosis .

1 is a schematic diagram illustrating a method for preparing a white Mugung flower extract according to the present invention.
Fig. 2 (A) is an image of white rosary flower; (B) is an image showing a 70% ethanol extract of white rosemary flower.
FIG. 3 (A) is a graph showing cell viability at low concentration treatment of white Mulberry flower extract; (B) is a graph showing TRAP activity.
FIG. 4 (A) is an image of TRAP staining of osteoclasts treated with a control group, white mugwort flower extract at a concentration of 50 μg / ml and 100 μg / ml, and (B) Lt; / RTI > (C) is a graph showing TRAP activity.
FIG. 5 is an image showing the evaluation of the genetic factors according to the treatment of white mugwort flower extract.

Hereinafter, the present invention will be described in more detail.

The reduction of osteoblast involved in osteogenesis or osteoclast-induced increase in bone mass leads to a decrease in bone mass and is associated with genetic and physiological disorders such as aging, hormone abnormality (estrogen deprivation), metabolic diseases and bone metastases As a preventive and remedy for osteoporosis caused by changes in environmental factors, there has been no disclosure about the pharmaceutical composition and the health functional food containing the extract of white mugwort flower as an effective ingredient.

Accordingly, the present invention provides a pharmaceutical composition for prevention and treatment of osteoporosis, which comprises a white mugwort flower extract as an active ingredient.

The White Hibiscus flowers (Hibiscus Syriacus L. ) is a deciduous broad-leaved shrub belonging to the genus Malvaceae. Its vagina is light, slightly fragrant, flowers are large and white is good, and the flavonoid component of the flavonoid is contained in a large amount in white mugwort. It is made of saponarin and has been used medicinally. In addition, it has been reported that the extract of Mugunghwa extract significantly inhibits the inflammatory mediator in the inflammation-induced macrophages and has anti-inflammatory activity.

In addition, according to the present invention, the white rose of Sharon flowers (flower of white Hibiscus syriacus, wHS ) in the extract vitro The present invention can provide a composition for preventing and treating osteoporosis because it can be applied to the control of osteoporosis.

At this time, TRAP activity may be decreased at a safety level concentration that does not affect the survival rate of the osteoclast treated with the composition. The osteoclast cell treated with the above composition exhibits cytotoxicity when the concentration of the composition is 250 ppm or higher, and the osteoclast survival rate may be remarkably lowered. However, the concentration of the white mugwort flower extract is 30-150 ppm, 120 ppm, the osteoclast treated with the above composition had cell stability and had a cell survival rate similar to that of the untreated group of white mugwort flower extract.

In addition, when the concentration of the above-mentioned white mugwort flower extract is 30 to 150 ppm, preferably 40 to 120 ppm, the osteoclast differentiation activity can be remarkably suppressed. More specifically, the concentration range is a concentration of the stability level that does not affect the survival rate of the osteoclast treated with the composition, and the TRAP activity of the osteoclast in the concentration range is 50-70 %.

In addition, the composition may decrease the expression of at least one selected from the group consisting of mRNA of Cal-R and mRNA of TRAP, and more particularly, the amount of Cal-R expression of osteoclast treated with the composition may be 50 ~ 55% lower than that of the untreated control group, and the amount of TRAP expression in osteoclasts can be reduced by 30 ~ 35% compared to the control group.

In addition, the pharmaceutical composition for prevention and treatment of osteoporosis, which comprises the extract of the present invention as an effective ingredient, effectively inhibits the differentiation of osteoclasts and thus is effective for preventing or improving osteoporosis. Accordingly, And provides a health functional food containing the product. When the composition according to the present invention is used as a food additive, the mixed extract may be directly added or used together with other food or food ingredients, and may be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to its intended use (prevention, health or therapeutic treatment).

There is no particular limitation on the kind of the food. Examples of the food to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include healthy foods in a conventional sense.

The health functional food of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Such natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention.

In addition to the above, the health functional food according to the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, , Alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the composition of the present invention may contain flesh for the production of natural fruit juice, fruit juice beverage and vegetable beverage. These components may be used independently or in combination. The ratio of such additives is not critical but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

In addition, the present invention provides a method for preparing a microorganism, which comprises a first step of washing white marigold flowers, drying and crushing them; Adding the solvent to the pulverized white mugwort flower at a volume ratio of 2 to 4; And extracting and cooling the extract, and concentrating the extract to prepare a white mugwort flower extract. The present invention also provides a method for preparing white mugwort flower extract for the prevention and treatment of osteoporosis. Hereinafter, the present invention will be described in more detail by step.

The method of the present invention for preparing ovalbumin flower for the prevention and treatment of osteoporosis according to the present invention is characterized in that the above step 1 is a step of washing white mugwort flowers and drying and crushing them, In order to increase the extraction yield.

The step 2 is a step of adding the solvent to the pulverized white mugwort flowers at a ratio of 2 to 4 times by volume. At this time, the method of extracting the white mugwort flower extract is not particularly limited as long as it is a method for extracting natural extracts, Water and at least one selected from the group consisting of C 1 to C 4 alcohols. More preferably water, ethanol or ethanol, but not particularly limited thereto.

In addition, the extraction in step 2 may be repeated at least twice at 80 ° C to 100 ° C for 1 to 5 hours, preferably at 80 ° C to 90 ° C for 3 to 5 hours, can do.

In step 3, the extract is cooled, filtered, and concentrated to prepare a white Mugung flower extract. Through this step, the extraction yield of white Mugung flower can be improved. At this time, the concentration in step 3 may be performed at 40 ° C to 50 ° C, preferably at about 45 ° C.

The yield of white mugwort flower extract obtained through the method of the present invention for preventing and treating osteoporosis according to the present invention may be 30% or more, preferably 30 to 35%.

Hereinafter, the present invention will be described in more detail with reference to the following examples. The following examples are provided to illustrate the present invention, but the scope of the present invention is not limited thereto.

[ Example ]

Example  1. Preparation of White Mugungwha Flower Extract

Mugunghwa was harvested in August, 2014, and was harvested in the area of Gunbuk-myeon, Geumsan-gun, Chungcheongnam-do. The white mugwort flowers were removed by impregnation using an ultrasonic washing machine (Branson, USA) before the experiment.

The sample from which the impurities were removed was dried in a freeze dryer (Martin Christ, Osterode, Germany), pulverized, and 3 times volume of 70% ethanol (EtOH) was added thereto. The mixture was refluxed 3 times at 85 ° C for 3 hours, After filtration using filter paper (No. 4), the mixture was concentrated under reduced pressure at 45 ° C by rotary evaporator (Rotavapor RII, Buchi, Switzerland). The extract was lyophilized and stored in a cryocooler in powder form Respectively. At this time, the final yield was 32.02%. The composition was diluted with PBS (Dulbecco's, phosphate buffered saline, Daegu, Korea) as shown in Table 1 to prepare a composition for treating and preventing osteoporosis according to the present invention.

Example The concentration of the composition
(Unit: ppm) Example 1 One Example 2 2.5 Example 3 5 Example 4 10 Example 5 25 Example 6 50 Example 7 100 Example 8 250 Example 9 500 Example 10 1000

Comparative Example  1. Control group

A solution of PBS (Dulbecco's, phosphate buffered saline, Daegu, Korea) not containing the above-mentioned white mugwort flower extract was prepared.

[ Experimental Example ]

All of the experimental groups described in the following Experimental Examples were performed in three or more groups, and the quantitative results were expressed as a percentage of the control group as the mean ± standard deviation. All experiments were repeated 3 times or more and used as the same test results. Two-tailed Student's t test was used to analyze the statistical significance, and p <0.05 was considered statistically significant.

Experimental Example  1. Measurement of osteoclast survival rate and evaluation of toxicity 1

MTT assay was performed to evaluate the survival and indirect toxicity of the composition of the present invention to osteoclasts. Mouse macrophage cell line RAW264.7 cells for induction of osteoclast differentiation were purchased from American Type Culture Collection (ATCC; Rockville, Md., USA) and cultured in RPMI 1640 supplemented with 10% FBS (fetal bovine serum) and 1% penicillin- streptomycin (100 U / Ml, 100 / / ml) in a 5% CO 2 , 37 ° C incubator.

The RAW 264.7 cells were seeded at a density of 1 × 10 4 cells / well in a 96-well plate and stabilized for 24 hours in DMEM with 10% FBS and 1% penicillin-streptomycin. 50 ng / ml of RANKL (PeproTech EC, London, England) was added to α-MEM (Gaithersburg, MD) containing 10% FBS and 1% penicillin-streptomycin for induction of osteoclast differentiation. The composition of Comparative Example 1 was prepared.

At that time, Comparative Example 1 was set as a control group. Three days after the incubation, the MTT solution [3- (4,5-dimethylthiazol-2-yl) -2,5- diphenyl tetrazolium bromide, Sigma-Aldrich] The reaction was carried out in a 5% CO 2 incubator at 37 ° C for 2 hours. The formazan crystals were dissolved in DMSO (dimethyl sulfoxide, Sigma-Aldrich). Thereafter, the absorbance was confirmed at 540 nm using an ELISA reader (BIO-RAD 450, California USA), and the results are shown in Table 2 and FIG.

Also, the TRAP solution assay was performed to measure the TRAP activity of the osteoclasts treated with the compositions of Examples 1 to 5 and Comparative Example 1 according to the present invention. The TRAP enzyme has high activity when ATP and nitrophenyl phosphate are present and can be used to confirm osteoclast differentiation level by measuring activity of osteoclast during bone resorption.

The cells were washed three times with PBS, fixed with 10% formalin for 5 minutes, and rinsed again with DW. Sigma TRAP staining (kit no. 387; Sigma-Aldrich, St. Louis, Mo., USA) was treated with fresh TRAP solution for 30 minutes according to instructions. The stained cells were observed with an optical microscope (CKX41, Olympus, Tokyo, Japan) and imaged with a digital imaging camera (DIXI 3000, Olympus, Tokyo, Japan) to obtain round-shaped osteoclast ), And the TRAP activity was measured. The results are shown in Table 2 and FIG. 3 below.

Comparative Example 1 Example 1 Example 2 Example 3 Example 4 Example 5 density 0 One 2.5 5 10 25 Cell survival rate 100 97.990 99.601 106.755 103.769 95.211 TRAP activity 100 95.953 91.624 101.511 97.772 87.807

According to Table 2 and FIG. 3, it was confirmed that Examples 1 to 5 showed cell survival similar to that of Comparative Example 1, and TRAP activity did not show a significant change. It was found that the low concentration of 0 ~ 30 ㎍ / ml of white Mulberry flower extract did not affect osteoclast differentiation inhibition.

Experimental Example  2. TRAP  Through dyeing Benign osteoclasts  Differentiation observation 2

The cell viability of the compositions of Examples 6 to 10 and Comparative Example 1 was measured in the same manner as in Experimental Example 1. The results are shown in Table 3 and FIG. The TRAP staining images of Example 6, Example 7 and Comparative Example 1 are shown in FIG. 4A, and the TRAP activity is shown in Table 4.

Comparative Example 1 Example 6 Example 7 Example 8 Example 9 Example 10 density 0 50 100 250 500 1000 Cell survival rate (%) 100 103.728 100.236 71.732 45.588 39.972

Comparative Example 1 Example 6 Example 7 density 0 50 100 TRAP activity (%) 100 47.276 39.738

According to Table 3 and FIG. 4, when the concentration of white Hibiscus flower extract was 50 and 100 / / ml, it was confirmed that the white Hibiscus flower extract did not exhibit cytotoxicity, similar to Comparative Example 1 in which the cell viability was the control group, When the concentration was 250 ~ 1000 ㎍ / ml, the cell survival rate was significantly decreased and it was confirmed that it shows cytotoxicity.

The TRAP activity of white Mugung flower extracts was 47.276% at 50 ㎍ / ml and 39.738% at 100 ㎍ / ml when the concentration of white Mugung flower extract was 50 and 100 ㎍ / ml, respectively .

Thus, in the case of a concentration of 30 to 150 占 퐂 / ml, preferably 40 to 120 占 퐂 / ml, in which the concentration of white Hibiscus flower extract is in the range of 50 and 100 占 퐂 / ml, The cell survival rate was improved by 0.1 ~ 5% and the TRAP activity was decreased by 50 ~ 70% compared to the untreated group.

Experimental Example  3. Reverse transcription polymerase chain reaction ( RT - PCR ) analysis

To examine the expression of calcitonin receptor (Cal-R) and TRAP in osteoclast treated with the composition of Example 7 and Comparative Example 1 according to the present invention, TRIzol was used to extract total RNA and iScript cDNA synthesis cDNA. The polymerase chain reaction (PCR) was carried out by using Emerald Taq for 10 min at 95 ° C for 1 min, followed by 1 min at 95 ° C, 30 sec at 51 ~ 58 ° C, and 1 min at 72 ° C After 25-32 cycles, final amplification was performed at 72 ° C for 10 minutes. The order of PCR primers used here is shown in Table 5 below. The reacted samples were electrophoresed on 2% agarose gel, stained with Et-Br, observed on UV, and quantified using densitometric analysis. The results are shown in FIG. 5 and Table 6 below.

gene The base sequence (5 '-> 3') Annealing temperature (캜) The size of the base sequence (bp) Cal-R
F-ACCGACGAGCAACGCCTACGC 58 490
R-GCCTTCACAGCCTTACAGGTAC TRAP
F-AAATCACTCTTTAAGACCAG 51
317
R-TTATTGAATAGCAGTGACAG GAPDH
F-AACTTTGGCATTGTGGAAG 53
223
R-ACACATTGGGGGTAGGAACA

Comparative Example 1 Example 7 density 0 100 Cal-R (%) 100 46.689 TRAP (%) 100 71.981

Osteoclasts are formed by complex processes such as initiation, differentiation, polynucleation, and maturation of various cytokines, hormones and genetic factors. There are abundant calcitonin receptors (Cal-R) and TRAP, , And it is known that it absorbs the bone matrix by forming an acting ring.

According to the results shown in Table 6 and FIG. 5, the mRNA expression levels of Cal-R and TRAP in the bone resorption step of the mature osteoclasts were analyzed by white rosemary flower extract, Cal-R expression was significantly reduced to 46.69% and TRAP expression was significantly reduced to 71.98% as compared with the control.

Therefore, it was found that the expression of Cal-R and TRAP mRNA was significantly inhibited by white Mugung flower extract as well as the inhibition of TRAP enzyme activity by RANKL. Thus, it can be seen that the composition containing the white mugwort flower extract of the concentration range can finally inhibit the absorption of the bone matrix.

Claims (10)

A pharmaceutical composition for the prevention and treatment of osteoporosis, which comprises an extract of white mugwort flowers as an active ingredient.
The pharmaceutical composition for the prevention and treatment of osteoporosis according to claim 1, wherein the concentration of the white mulberry flower extract is 30 to 150 ppm.
3. The method of claim 2,
Wherein the concentration range is a concentration of a stability level that does not affect the survival rate of the osteoclast treated with the composition, and the TRAP activity of the osteoclast is decreased in the concentration range.
The method according to claim 1,
Wherein said composition reduces the expression of at least one selected from the group consisting of Cal-R mRNA and TRAP mRNA.
A health functional food for preventing and improving osteoporosis comprising the composition of any one of claims 1 to 4.
A step of washing white Mugung flower, drying and crushing it;
Adding the solvent to the pulverized white mugwort flower at a volume ratio of 2 to 4; And
Cooling, filtering and concentrating the extract to prepare white mugwort flower extract;
Wherein the method comprises the steps of:
The method according to claim 6,
Wherein the solvent comprises at least one member selected from the group consisting of water and C 1 -C 4 alcohols.
The method according to claim 6,
Wherein the extract is carried out at 80 ° C to 100 ° C for at least 2 or more times for 1 to 5 hours.
The method according to claim 6,
Wherein the concentration is carried out at 40 ° C to 50 ° C in the step 3, and a method for the preparation of a white Mugung flower extract for the prevention and treatment of osteoporosis.
The method according to claim 6,
Wherein the yield of the white mugwort flower extract is 30% to 35%.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20200084624A (en) * 2019-01-03 2020-07-13 숙명여자대학교산학협력단 Chocolate containing mugunghwa powder and manufacturing method thereof
KR20210066384A (en) * 2019-11-28 2021-06-07 숙명여자대학교산학협력단 Hibiscus syriacus l. agent for food and manufacturing method of the same
KR20220064011A (en) * 2020-11-11 2022-05-18 대한민국(산림청 국립산림과학원장) Composition for prevention and treatment of osteoporosis containing extracts of Jeoktanshim group of Hibiscus syriacus as an active ingredient

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20200084624A (en) * 2019-01-03 2020-07-13 숙명여자대학교산학협력단 Chocolate containing mugunghwa powder and manufacturing method thereof
KR20210066384A (en) * 2019-11-28 2021-06-07 숙명여자대학교산학협력단 Hibiscus syriacus l. agent for food and manufacturing method of the same
KR20220064011A (en) * 2020-11-11 2022-05-18 대한민국(산림청 국립산림과학원장) Composition for prevention and treatment of osteoporosis containing extracts of Jeoktanshim group of Hibiscus syriacus as an active ingredient

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