KR20180002141A - Food composition for improvement of osteoporosis with the extract of barley sprouts and pharmaceutic compositon for prevention and treatment of osteoporosis with the extract of Hordeum vulgare - Google Patents

Abstract

The present invention relates to a food composition for alleviating osteoporosis and a pharmaceutic composition for the prevention and treatment of osteoporosis containing a hot water extract of barley sprouts, wherein the hot water extract of barley sprouts can alleviate, prevent, or treat osteoporosis by lowering the activity of tartrate-resistant acid phosphatase (TRAP) in bone tissues and increasing the activity of alkaline phosphatase (ALP).

Description

TECHNICAL FIELD The present invention relates to a food composition for improving osteoporosis and a method for preventing or treating osteoporosis. The present invention relates to a food composition for preventing osteoporosis,

The present invention relates to a food composition for osteoporosis improvement comprising a hot spring extract of barley barley, and a pharmaceutical composition for preventing or treating osteoporosis.

Bone remodeling continues after bone growth, and the homeostasis between bone formation by osteoblasts and bone resorption by osteoclasts is maintained through this process, Tissue has a balance of bone formation and bone resorption [Refs. 1, 2].

Osteoporosis is a disease that easily causes fracture of the radial, thigh, and spine due to a change in the composition of the skeleton due to the imbalance of calcium metabolism, resulting in a decrease in bone mass. . Especially, postmenopausal women have the highest incidence of estrogen secretion, which has been reported as a major cause [Ref. 3, 4].

Osteoblasts differentiated by various factors (BMP, ALP, TGF-β) secrete non-protein and protein components and fully differentiated osteoblasts express osteocytes within the bone matrix ). Differentiation into osteoclasts is a very important process for bone resorption. The main signal mediator that mediates differentiation is RANKL (receptor activator of NF-κB ligand), which binds to RANK expressed on the surface of osteoclasts and plays an essential role in osteoclast differentiation Induces the expression of a substance such as NF-κB, c-FOS [Refs. 5-7]

Estrogen therapy, calcitonin therapy, calcium supplements, and vitamin D derivative therapy have been known to treat osteoporosis. However, estrogen therapy and calcitonin therapy have been reported to be the most frequently used methods. However, it has been reported that it is difficult to control various side effects and timing of administration [Refs. 8, 9].

On the other hand, when the bud of barley is a young leaf of barley and the light of low energy is received, the production of C-glycosylflavone is increased and a lot of it accumulates in the pigment. Especially, 6-c-glycosyl-7-o- (2'-O-glycosylisovitexin) is known to be a typical flavonoid antioxidant found in barley leaves [6-c-glycosyl-7-o-glucosylapigenin] . It has already been proved that there is a functional substance which has the effect of preventing and improving hyperlipidemia and diabetes [Reference 10].

Korean Patent Laid-open Publication No. 10-2016-0006368 (published on January 19, 2016) describes "a composition for preventing and treating osteoporosis, comprising a mixture of bokbunja and hwanggi group".

[Reference 1] Raisz LG. Hormonal regulation of bone growth and remodeling. In Cell and Molecular Biology of Vertebrate Hard Tissue CIBA Foundation Symposium 136, pp. 226-238, Wiley, NY, USA (1988) References [2] Lee. YS. Effect of isoflavones on proliferation and oxidative stress of MC3T3-E1 osteoblast-like cells. Korea soybean Digest. 18: 35-42 (2001) [Reference 3] Jilka RL. Cytokines, bone remodeling and estrogen deficiency. Bone 23: 75-81 (1998) [Reference 4] Udagawa N, Takakashi N, Akatsu T, Tanaka H, Sasaki T. Nishihara T. Origin of osteoclasts: mature monocytes and macrophages are capable of differentiating into osteoclasts under a suitable microenvironment prepared by bone marrow-derived stromal cells. Proc Natl Acad Sci USA 87: 7260-7264 (1990) [Reference 5] Milsicek RJ. Interaction of naturally occurring nonsteroidal estrogen with expressed recombinant human estrogen receptor. J Streoid Biochem Mol Biol 49: 153-460 (1994) [Reference 6] Almeida M, Han L, Martin-Millan M, Plotkin Li, Stewart SA, Roberson PK, Kousteni S, O'Brience, Bellid T, Parfitt AM, Weinstein RS, Jilka RL, Manolagas SC. Skeletal involution by age-associated oxidative stress and its acceleration by sex steroids. J Biol Chem 282: 27285-27297 (2007) [Reference 7] Maeda T, Matsunuma A, Kawane T, Horiuchi N. Simavastatin promotes osteoblast differentiation and mineralization in MC3T3-E1 cells. Biochem Biophys Res Commun. 283: 874-877 (2001) [Reference 8] Calck AP, Schuttinga JA, Targeted estrogen / proestrogen replacement therapy for osteoporosis. Calculation of health care cost savings. Osteoporos Int 2: 195-200 (1992) [Reference 9] Holland EF, Studd JW, Mansell JP. Changes in collagen composition and cross-links in osteoporotic postmenopausal women treated wit percutaneous estradiol implant Obstet Gynecol 83: 180-183 (1994) [Reference 10] Byun AR, Chun HJ, Lee J, Lee SW, Lee HS, Shim KW. Effect of a dietary supplement with Barley sprout extract on blood cholesterol metabolism. 473056, pp.7 (2015)

In the present invention, prophylactic and improving efficacy of osteoporosis of the barley extract is demonstrated, and it is developed and provided as a functional material through the study of the mechanism.

The present invention provides a food composition for improving osteoporosis characterized by containing a hot spring water extract of barley barley as an active ingredient.

The present invention contains a hot-water extract of sprout barley as an active ingredient. The sprout barley is a young leaf of barley whose scientific name is Hordeum vulgare to be. In the present invention, it has been found that osteoporosis is effective for improving the hot-water extract of barley barley, and the present invention has been completed.

The food composition of the present invention is meant to include any food containing the hot spring water extract of rosemary, and the ingestion of the food composition results in the improvement of osteoporosis. Specifically, it may be directly referred to as a powder state in which powder of barley hot-water extract is pulverized, and examples thereof include forms added to various processed foods including beverages. The constructions are not particularly limited, and include any of solid, semi-solid, gel, liquid, and powder.

In the food composition for osteoporosis improvement according to the present invention, the hot-water extract of bud of barley preferably lowers the activity of TRAP (tartrate-resistant acid phosphatase) and enhances the activity of ALP (alkaline phosphatase) have.

On the other hand, the present invention provides a pharmaceutical composition for preventing or treating osteoporosis characterized by containing a hot spring water extract of bud of barley as an active ingredient.

When the composition for the prevention or treatment of osteoporosis of the present invention is used clinically, it can be used together with a carrier commonly used in the pharmaceutical field, and can be used in the form of a conventional preparation (for example, tablets, capsules, A liquid preparation, a suspension, and the like).

The pharmaceutical compositions of the present invention may be administered orally or parenterally, for example, intravenously, subcutaneously, intraperitoneally or topically. The dosage of the composition for the prevention or treatment of osteoporosis of the present invention may vary from 0.01 mg / kg to 10 g / kg per day, preferably from 1 mg / kg to 1 g / kg per day ≪ / RTI > Generally, it is administered to an adult in an amount of 10 to 500 mg, preferably 50 to 300 mg per day, divided into several times a day, preferably one to six times, at regular intervals according to the judgment of a doctor or pharmacist Lt; / RTI >

In the pharmaceutical composition for the prevention or treatment of osteoporosis according to the present invention, the hot-boiled hot- It may be possible to lower the activity of the tartrate-resistant acid phosphatase (TRAP) enzyme in the bone tissue and increase the activity of the alkaline phosphatase (ALP) enzyme.

The hot spring water extract of the present invention can improve, prevent or treat osteoporosis by lowering the activity of tartrate-resistant acid phosphatase (TRAP) enzyme in bone tissue and increasing the activity of ALP (alkaline phosphatase) enzyme.

FIG. 1 is a graph showing the effect of the present invention on the degree of differentiation of osteoclasts.
FIG. 2 is a graph showing the effect of the present invention on the activity of alkaline phosphatase (ALP).
3 is a primer sequence used for measuring the expression of the gene.
FIG. 4 is a graph showing the effect of the present invention on the expression of RANKL gene.
FIG. 5 is a graph showing the effect of the present invention on the expression of ALP gene.
FIG. 6 is a graph showing the effect of the extract of the present invention on the expression of IGF-1 gene. FIG.
FIG. 7 is a graph showing the effect of the present invention on the expression of an estrogen receptor gene.
FIG. 8 is a graph showing the effect of the extract of bud of the present invention on osteocalcin gene expression.

Hereinafter, the present invention will be described in more detail with reference to the following Examples and Experimental Examples. However, the scope of the present invention is not limited to the following examples and experimental examples, and includes modifications of equivalent technical ideas.

[ Example  1: Preparation of barberry extract of buds]

The shoot barley containing harvested seeds and sprouts was cut into pieces of 2 cm or less by using a rotary slicer and hot-air-dried at 90 ° C for 8 hours with hot water extraction (20 times, 3.1 brix) , Concentrated under reduced pressure, lyophilized and pulverized (100 mesh).

Pilot production conditions of shoot barley, final yield and Birx concentration Raw material input
(kg) Quantity of water
(L) Heating time
(hour) Temperature
(° C) Final yield
(%) Brix Remarks Sprouts barley 10 200 8 90.2 78.5 3.01 157L

[ Example 2: TRAP ( tartrate -resistant acid 포스화제 ) Measure]

In this Example, an experiment was conducted in order to confirm the effect of the present invention on the degree of differentiation of osteoclasts.

1 × 10 ⁵ RAW 264.7 osteoclast precursor cells were seeded in 6-well plates and cultured for 3 days in conditions of osteoblast cultures treated with 30 ng / mL of RANKL and shoot barley extract. 50 mM citrate buffer containing 1.36 mg / mL 4-nitrophenyl phosphate disodium salt and 10 mM tartrate was prepared as the substrate solution.

After incubation at 37 ° C in a 5% CO ₂ incubator for 30 min, the reaction mixture was stopped with 0.1 N NaOH and the optical density at 405 nm was measured three times And an average value and a standard deviation thereof were determined.

The activity of TRAP enzyme increases when osteoclasts undergo bone resorption. After formation of monoclonal osteoclast precursor cells, the osteoclast cell is formed, where the cells are fused, and adheres to the bone surface. In addition, TRAP is an osteolytic enzyme that is active only in the presence of nitrophenyl phosphate and ATP, and is the only osteolytic enzyme that exists only in osteoclasts.

As a result, TRAP was significantly increased in osteoclast differentiation induced osteoclast differentiation, osteoclast differentiation inducing cells were treated with 100, 200, 400 When treated with μg / mL, TRAP decreased by 6.77%, 20.83%, and 35.36%, respectively, compared with the control group without the bud. Especially, it was confirmed that the treatment of barley extracts at 200, 400 μg / mL decreased significantly ( P <0.05) (FIG. 1). FIG. 1 is a graph showing the effect of the present invention on the degree of differentiation of osteoclasts.

[ Example  3: Alkaline  Phosphatase (Alkaline 포스화제 ) Active measurement]

In this experiment, an experiment was conducted to confirm the effect of the extract of the present invention on alkaline phosphatase activity.

5 × 10 ³ SaOs-2 cells were seeded in 6-well plates and 50 μg / mL ascorbic acid and 10 mM β-glycerophosphate were added to induce osteoblast differentiation Respectively. After incubation at 37 ° C in a CO ₂ incubator at 37 ° C for 48 hours, the cells were washed twice with PBS, 0.2% triton X-100 was added, and lysed in a 37 ° C incubator for 30 minutes . After centrifugation at 2,500 rpm for 10 minutes, the supernatant was taken to quantify the protein. The remaining supernatant was added with 0.1 N glycine and 100 mM p-NPP (p-nitrophenylphosphate) and incubated at 37 ° C for 30 minutes. N NaOH was added to stop the reaction, and the absorbance was measured at 405 nm to calculate the amount converted to p-nitrophenol by ALP enzyme.

Alkaline phosphatase (basic phosphatase) is present in most tissues, and ALP in bone tissue is known to increase its activity when bone growth occurs actively.

(100, 200, and 400 μg / mL), respectively, showed that ALP activity was 113.54%, 225.79%, and 285.73%, respectively, compared to the untreated group. ( P < 0.05) (Fig. 2). Fig. 2 is a graph showing the effect of the extract of bud of the present invention on ALP activity.

[ Example  4: Expression of osteoclast differentiation factor in osteoblast cells]

Real time PCR was performed to investigate the expression of osteoclast differentiation factors in osteoblasts when treated with barley extract. 5 × 10 ³ SaOs-2 cells were seeded in 6-well plates and 50 μg / mL ascorbic acid and 10 mM β-glycerophosphate were added to induce osteoblast differentiation After 48 hours of treatment, the cells were harvested and RNA extraction was performed using the RNeasy extraction kit (Qiagen, Gaithersburg, Maryland, USA) according to the manufacturer's protocol. cDNA was synthesized using iScript cDNA synthesis kit (Bio-Rad Laboratories Headquarters, Hercules, Calif., USA).

Real-time PCR (Applied Biosystems, Foster City, CA, USA) was performed using SYBR Green (iQ SYBR Green Supermix, Bio-Rad Bio-Rad Laboratories Inc.) ) Were used.

The primer sequence used to measure the expression of a specific gene is shown in Fig. In real-time PCR reactions, 2 μL of cDNA and 10 μL of 2X SYBR mix were added to the total 20 μL, and 1 μL of 100 pmol / μL of the forward / reverse primer was added, respectively, and the remainder was filled with H ₂ O. The PCR amplification step was as follows and the amplification cycle was 40 cycles. Denaturation at 95 ° C for 15 seconds, annealing at 55 ° C for 30 seconds and extension at 72 ° C for 8 minutes at 95 ° C for hot start, denaturation at 95 ° C for 15 seconds, Repeated for 30 seconds, and the value after each cycle was recorded. Melting curve analysis was performed to confirm the specificity of the primer after all cycles were completed. Analysis of the results was performed with One step system software v2.1 provided by Applied Biosystems.

Osteoclast differentiation is initiated by the binding of RANKL (receptor activator of nuclear facotr-κB ligand), an osteoclast differentiation inducer expressed in osteoblasts, and its receptor RANK expressed in osteoclast precursor cells. The expression of RANKL gene was 4.68% and 20.07%, respectively, compared with the untreated group (100, 200, and 400 μg / mL) And 41.6%, respectively. 200, and 400 μg / mL, respectively ( P <0.05) (Fig. 4). FIG. 4 is a graph showing the effect of the present invention on the expression of RANKL gene.

The expression of ALP gene was increased by 100.88%, 134.36%, and 185.46%, respectively, as compared with the untreated group. ( P <0.05) in the group treated with 200, 400 μg / mL of barley extract (FIG. 5). FIG. 5 is a graph showing the effect of the present invention on the expression of ALP gene.

IGF-1 plays an important role in bone metabolism, increasing the transcription of type I collagen (type Ⅰ collagen) and inhibiting the transcription of collagen protease, matrix metalloproteinase . The expression of IGF-1 gene was increased by 104.6%, 115.9% and 154.1%, respectively, compared with the untreated group. However, the expression level of IGF-1 gene was increased by 154.1% ( P <0.05) only at 400 μg / mL (FIG. 6). FIG. 6 is a graph showing the effect of the extract of the present invention on the expression of IGF-1 gene. FIG.

Estrogen, a female hormone, has been reported to regulate the differentiation of osteoclasts after binding to receptors present on the cell membrane or nuclear membrane. The expression of estrogen receptor gene was increased by 104.4%, 132.1%, and 169.5%, respectively, compared with the untreated group. Respectively. ( P <0.05) in the group treated with 200 μg / ml of bud of barley extract (FIG. 7). FIG. 7 is a graph showing the effect of the present invention on the expression of an estrogen receptor gene.

Osteocalcin is a vitamin K-dependent α-carboxyglutamic acid protein that binds to calcium and binds to hydroxyapatite, a typical mineral of bone, and is a very specific protein for bone and dentin. The expression of osteocalcin gene was 179.2% in the group treated with 200, 400 μg / mL compared to the group not treated with the sample, , And 193.7%, respectively ( P < 0.05) (Fig. 8). FIG. 8 is a graph showing the effect of the extract of bud of the present invention on osteocalcin gene expression.

Claims (4)

A food composition for improving osteoporosis characterized by containing hot-water extract of barley barley as an active ingredient
The method according to claim 1,
The dried barley hot-water extract,
Wherein the activity of TRAP (tartrate-resistant acid phosphatase) enzyme is lowered and the activity of ALP (alkaline phosphatase) enzyme is increased in bone tissue.
A pharmaceutical composition for preventing or treating osteoporosis characterized by containing hot-water extract of barley barley as an active ingredient.
The method of claim 3,
The dried barley hot-water extract,
Wherein the activity of TRAP (tartrate-resistant acid phosphatase) enzyme is lowered and the activity of ALP (alkaline phosphatase) enzyme is increased in bone tissue.

Images