WO2014200159A1 - Composition for promoting osteogenesis and bone growth comprising hwanggeumchal sorghum extract as active ingredient - Google Patents

Abstract

The present invention relates to a composition for osteogenesis and bone growth comprising a hwanggeumchal sorghum extract as an active ingredient.

Description

A composition for promoting bone formation and bone growth comprising the extract of Golden Stalk Water as an active ingredient

The present invention relates to a composition for bone formation and bone growth comprising the golden wax water extract as an active ingredient.

In general, the most important for postnatal vertical (vertical) bone growth is insulin-like growth factor-1 (IGF-1) and growth hormone (GH). These growth-related hormones come from the pituitary gland and when they reach the liver cells, they activate the insulin-like growth factor gene called IGF-1. This IGF-1 is delivered to cartilage and bones, increasing in height. People with defects in the IGF-1 gene are known to be unable to grow properly during pregnancy or to become retarded children after birth. Providing growth hormone to children whose hormones are not secreted at all in the brain, or by supplying substances that activate genes of growth hormone, will of course help the key growth. But not all children are deficient in the hormones they supply. There is enough hormone in the brain, but in the process of supplying it, the growth of height can be slowed even if other organs do not properly convert it to IGF-1. In other words, if IGF-1 is activated and increased in the osteoblasts (osteoblasts) that make bones that do not supply enough hormones in the brain, the cartilage and bones will have sufficient conditions for height growth.

Insulin-like growth factor-1 receptor (IGF-IR) is a transmembrane receptor tyrosine kinase that is activated by insulin and ligands of IGF-I and IGF-II, and mice lacking this IGF-1R gene (KO mice; knock out mice) is a potent growth-related protein that causes death of respiratory failure shortly after birth due to poor development of respiratory muscles. However, there are transcriptional regulatory proteins that bind to the IGF-1 and IGF-1R genes and the promoter sites of these two genes in the cell's nucleus, which are STAT5 (signal transducer and activator of transcription 5) proteins. . Recently, the function of this STAT5 protein is becoming increasingly important. In other words, kinase enzymes such as Jak2 play an important role in activating and recruiting STAT5 (or STAT5b).

Therefore, finding a variety of methods or drugs (HSEs) that can regulate or increase these STAT5 proteins with these key functions will help you grow and grow your bones.

[Previous Patent Document]

Korean Patent Publication No. 10-2012-0021389

Korean Patent Publication No. 10-2012-0097294

The present invention has been made in view of the above necessity, and an object of the present invention is to prepare a composition which helps to activate bone, grow and grow bone by participating in the proliferation of osteoblasts and activation of growth factors.

In order to achieve the above object, the present invention provides a pharmaceutical composition for promoting bone formation and bone growth comprising the extract of golden wax water as an active ingredient.

In one embodiment of the invention, the extract is preferably an alcohol extract, the alcohol is more preferably methanol, ethanol, propanol or butanol, in the embodiment of the present invention used methanol.

In another aspect, the present invention provides a food composition for improving bone formation and bone growth comprising the extract of golden wax water as an active ingredient.

In another aspect, the present invention is a group consisting of insulin-like growth factor (IGF) -1 receptor, growth hormone (GH) receptor, and bone morphogenetic protein (BMP) 7 containing golden glutinous water extract as an active ingredient It provides a composition for promoting at least one protein selected from.

In another aspect, the present invention provides a composition for promoting the expression of signal transducer and activator of transcription (STAT) 5b protein containing golden extract extract as an active ingredient.

In another aspect, the present invention is treated with the extract of golden wax water on the cells insulin-like growth factor (IGF) -1, growth hormone (GH) receptor, bone morphogenetic protein (BMP) 7 or It provides a method for promoting signal transducer and activator of transcription (STAT) 5b protein expression.

* The pharmaceutical composition for promoting bone formation and bone growth of the present invention contains the extract in an amount of 0.1 to 50% by weight based on the total weight of the composition.

Pharmaceutical compositions comprising the extract of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Pharmaceutical dosage forms of the extracts of the present invention may be used in the form of their pharmaceutically acceptable salts, or may be used alone or in combination with other pharmaceutically active compounds, as well as in a suitable collection.

It can be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like oral formulations, external preparations, suppositories, and sterile injectable solutions. Carriers, excipients and diluents that may be included in compositions comprising wood vinegar include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations include at least one excipient such as starch, calcium carbonate, sucrose in the extract. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the extracts of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the extract of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably 0.001 to 100 mg / kg per day. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

The extract of the present invention can be administered to mammals such as mice, mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

Since the extract of the present invention has little toxicity and side effects, it is a drug that can be used safely even when taken for a long time.

The present invention provides a dietary supplement comprising a golden corn bran extract and a food acceptable acceptable food supplement additive exhibiting the effect of promoting bone growth. Foods to which the extract can be added include, for example, various foods, beverages, gums, teas, vitamin complexes, health supplements, and the like, and can be used in the form of powders, granules, tablets, capsules, or beverages.

It may also be added to foods or beverages for the purpose of promoting bone growth. At this time, the amount of the extract in the food or beverage can be added in 0.01 to 15% by weight of the total food weight, the health beverage composition is added in a ratio of 0.02 to 5 g, preferably 0.3 to 1 g based on 100 ml Can be.

The health functional beverage composition of the present invention is not particularly limited to other ingredients except for containing the extract as an essential ingredient in the indicated proportions, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the extract of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the extract of the present invention may contain natural fruit juice and fruit flesh for the production of fruit juice beverages and vegetable beverages.

These components can be used independently or in combination. The proportion of such additives is not so critical but is usually selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the extract of the present invention.

Hereinafter, the present invention will be described.

The present inventors have completed the present invention as a result of making an effort to prepare a composition that helps to increase bone growth and growth by regulating or increasing STAT5 protein, thereby participating in osteoblast proliferation and activation of growth factors.

The present invention relates to a composition for activating bone growth of golden wax water (HSE), and to a composition for activating bone growth, comprising a pharmaceutically acceptable additive.

The composition of the present invention can regulate or activate Jak2 and STAT5b proteins that mediate expression of IGF-IR, IGF-1 and GHR genes that contribute to bone growth in osteoblasts (osteoblasts) of humans and animals. In addition, STAT5b is deeply involved in the regulation of BMP7 protein, which is important for osteoblast differentiation in bone growth. The Jak2 and STAT5b-mediated bone growth is activated to increase the height of growing children.

The composition of the present invention can modulate or increase the STAT5 (or STAT5b) protein, which mediates IGF-I, IGF-IR and GHR gene and protein expression, which contributes to bone growth and height, thereby mediating the STAT5 It has the effect of bone activity, bone growth and growth.

1 shows the effect of HSE on survival in MC3T3-E1 cells. MC3T3-E1 cells were incubated for 24 hours in 96-well dishes and then treated with HSE at the concentrations described for 24 hours. After incubation, cell viability was assessed using an MTT assay. Data is representative of three independent experiments.

2-4 show the effect of HSE on the expression of growth hormone signaling related proteins in MC3T3-E1 cells. (FIG. 2) MC3T3-E1 cells were treated for 24 hours with increasing concentrations of HSE. (FIG. 3) Cells treated with HSE for 24 hours after 4 hours without or pretreated with 50 μM AG490. Protein extract (20 μg) isolated by 10% SDS-PAGE and western blot was performed. Beta-actin was used as protein loading control. (Figure 4) Relative levels of BMP7, GHR and Jak2 proteins were determined using densitometry analysis and normalized to the amount of beta-actin. This image is representative of three independent experiments. Asterisks indicate a statistically significant increase or decrease by ANOVA (*** p <0.001).

5 to 8 show that HSE activates expression of growth hormone signaling mRNA in MC3T3-E1 cells. Total RNA was isolated from MC3T3-E1 cells using the RNeasy kit. Amplify cDNA using specific primers for BPM7, growth hormone receptor (GHR) or 18S. 18S was used as loading control. (FIG. 5) MC3T3-E1 cells were treated with the indicated concentrations of HSE for 24 hours. (Figure 6) Relative levels of BMP7 and GHR mRNAs were determined using densitometer analysis and normalized to 18S amounts. (FIG. 7) MC3T3-E1 cells were treated with 50 μM AG490 for 4 hours before or without treatment for 24 hours with HSE. (FIG. 8) Relative levels of BMP7 and GHR mRNAs were determined using densitometer analysis and normalized to 18S amount. Data shown are representative of three independent experiments. Asterisks indicate a statistically significant increase or decrease by ANOVA (*** p <0.001).

9-10 show that HSE-increased BMP7 and GH signaling requires STAT5b activation in MC3T3-E1 cells. (FIG. 9) MC3T3-E1 cells were grown to 50% confluence and then transfected with ON-TARGETplus Non-targeting siRNA or ON-TARGETplus SMARTpool siRNA targeting STAT5b using FuGene 6 according to the manufacturer's instructions. After 48 hours of transfection, cells were incubated in serum-free medium for 24 hours and then incubated for 24 hours with 30 μg / ml HSE. Protein extract (20 μg) was separated by 10% SDS-PAGE and western blot was performed. Beta-actin used as protein loading control. (FIG. 10) Relative levels of STAT5b, p-STAT5b, BMP7 and IGF-1R proteins were determined using densitometer analysis and normalized to the amount of beta-actin. Data shown are representative of three independent experiments. Asterisks indicate a statistically significant increase or decrease by t-test (** p <0.01, *** p <0.001).

Hereinafter, the present invention will be described in more detail with reference to non-limiting examples. However, the following examples are intended to illustrate the invention and the scope of the present invention is not to be construed as limited by the following examples.

Example 1 Extraction and Separation of HSE

In the present invention, Hwanggeumchal sorghum was selected. The HS was extracted with a slight improvement of Chung and Kim methods (Chung IM and Kim SH: Analysis methods of phenol compounds.Korean Society of Crop Science 8: 5-12, 2004). Samples (50 g) were kept at -36 ° C and ground before use. The ground powder was extracted with 30% 0.1 N HCl in acetonitrile. The solution was filtered (Whatman No. 42), concentrated and extracted again with 100% methanol and dried overnight with lyophilizer. The waxy water extract was characterized by its phenolic component using HPLC.

Example 2: Cell Culture

MC3T3-E1 osteoblasts were cultured in alpha-MEM medium (Sigma Chemical, USA) containing 10% fetal bovine serum (FBS) and 100 U / ml penicillin.

The cultured cells were resuspended in a suitable medium at a density of 2.5 × 10 ⁵ cells / ml for each experiment.

Example 3 Investigation of the Effect on the Viability of Osteoblasts by HSE

The present invention first treated the HSE according to the concentration (0, 10, 20 and 40 μg / ml) using MC3T3-E1 cell line in 96-well plates, incubated for 24 hours and then active only on the living cells After treatment with blue formazan (MTT) and incubated for 4 hours at 37 ℃. Cells were disrupted and cell lysate was obtained. Formazan products are dissolved at each well by DMSO and read at 550 nm. The procedure was repeated three times and representative results are shown in FIG. 1.

Example 4 Investigation of the Effect of HSE on the Expression of GH Signaling-Related Proteins and BMP7 Protein

In order to investigate the effect of HSE on the expression of growth hormone signaling proteins and BMP7 protein, the following experiments were performed.

Treatment of HSE according to concentration (0, 10, 20 and 40 μg / ml) using MC3T3-E1 cell line or pretreatment with Jak2 inhibitor AG490 followed by incubation for 24 hours with 30 μg / ml HSE Incubated for the expression of each protein regulated by HSE.

That is, the cell lysate obtained by adding RIPA lysis buffer (Gibco-BRL, USA) to each of the cultured cells was developed on a 10% SDS-polyacrylamide gel and transferred again on a nitrocellulose membrane, followed by BMP7, STAT5b, Blots were used with antibodies against p-STAT5, IGF-1R, GHR and Jak2 proteins and labeled with anti-β-actin antibody (FIG. 2). As shown in FIG. 2, the protein levels of BMP7, STAT5b, p-STAT5, IGF-1R, GHR and Jak2 were dose- and concentration-dependently up-regulated by HSE. In addition, as shown in Figures 2 to 4, the treatment of HSE, the protein expression of BMP7, GHR and Jak2 increased, pretreatment with AG490 was found to decrease the expression of BMP7, GHR and Jak2 protein.

Example 5 Investigation of the Effect of HSE on BMP7, GHR and Jak2 Gene Expression

Total RNA semi-quantitative analysis using RT-PCR was performed to investigate the effects of HSE-stimulated MC3T3-E1 on the transcriptional activity of BMP7, GHR and Jak2.

First, MC3T3-E1 was used to treat HSE according to the concentration (0, 10, 20 and 40 μg / ml) and incubated for 24 hours, or pretreated with Jak2 inhibitor AG490 and then treated with 30 μg / ml HSE. The transcription mRNAs of BMP7, GHR and Jak2 genes were determined by real-time polymerase chain reaction (PCR) and Western blot. Each measurement was obtained by repeating the measurement three times by normalizing the β-actin mRNAs level. As a result, as shown in Figures 5 to 8, the treatment of HSE, BMP7, GHR and Jak2 gene expression was increased, and AG490 pretreatment was found to decrease the BMP7, GHR and Jak2 gene expression. Therefore, it can be seen that signaling induced by HSE is regulated similarly to GH signaling through Jak2 / STAT5b (FIGS. 5 to 8).

Example 6 Investigation of the Effect of STAT5b Protein on BMP7 and GH Signaling Increased by HSE

    To determine whether STAT5b protein is activated by sorghum extract in osteoblasts (MC3T3-E1), the expression levels of STAT5b, p-STAT5b, BMP-7 and IGF-1R were examined using STAT5b siRNA.

    When MC3T3-E1 cell line is about 50% grown, transfection of ON-TARGETplus SMARTpool siRNA targeting STAT5b or ON-TARGETplus Non-targeting siRNA with FuGene 6 is followed by incubation for 24 hours after switching to serum free medium for 48 hours. After treatment with μg / ml HSE and incubated for 24 hours, proteins were extracted to investigate whether STAT5b was involved in signaling by HSE.

    That is, the cell lysate obtained by adding RIPA lysis buffer (Gibco-BRL, USA) to each of the cultured cells was run on a 10% SDS-polyacrylamide gel and again transferred onto a nitrocellulose membrane, followed by STAT5b, p- Blots using antibodies against STAT5, BMP7 and IGF-1R proteins and labeled using anti-β-actin antibody (FIG. 4). As a result, as shown in FIGS. 9 to 10, the protein expression levels of STAT5b, p-STAT5b, BMP-7 and IGF-1R, which were increased by HSE, were treated with HSTAT after silencing with siSTAT5b, resulting in STAT5b, p-STAT5b, and BMP. -7 and IGF-1R gene was confirmed to be silenced.

Having described the specific part of the present invention in detail, it will be apparent to those of ordinary skill in the art that such a specific description is merely a preferred embodiment, thereby not limiting the scope of the present invention. will be. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (9)

1. A pharmaceutical composition for promoting bone formation and bone growth, comprising the extract of golden wax water as an active ingredient.
2. The pharmaceutical composition of claim 1, wherein the extract is an alcohol extract.
3. The pharmaceutical composition of claim 2, wherein the alcohol is methanol, ethanol, propanol or butanol.
4. Food composition for bone formation and bone growth improvement comprising the golden wax water extract as an active ingredient.
5. The food composition of claim 4, wherein the extract is an alcohol extract.
6. 6. A food composition according to claim 5, wherein the alcohol is methanol, ethanol, propanol or butanol.
7. At least one selected from the group consisting of insulin-like growth factor (IGF) -1 receptor, growth hormone (GH) receptor, and bone morphogenetic protein (BMP) 7, including golden sorghum extract as an active ingredient Protein expression promoting composition.
8. STAT (signal transducer and activator of transcription) 5b protein expression promoting composition comprising the extract of golden water.
9. By treating the cells with golden extract, the insulin-like growth factor (IGF) -1, growth hormone (GH) receptor, bone morphogenetic protein (BMP) 7, or STAT (signal transducer) of the cells. and activator of transcription).

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