WO2019208913A1 - Composition comprising sanguisorba officinalis l. extract as effective ingredient for promoting bone growth - Google Patents

Abstract

The present invention relates to a composition comprising a Sanguisorba officinalis L. extract as an effective ingredient for promoting bone growth. When fed as medicaments or foods to children and adolescents, who are in the growth period in their lives, the bone growth promoting composition comprising a Sanguisorba officinalis L. extract of the present invention exhibits the effects of increasing bone length and expanding the physes to promote bone growth as well as maintaining or increasing bone density in children and adolescents.

Description

Bone growth promoting composition comprising fat as an active ingredient

The present invention relates to a composition for promoting bone growth comprising Sanguisorba officinalis L. extract as an active ingredient, more specifically, pharmaceutical or food that can promote bone growth by administering or ingesting children or adolescents in the growing phase. It relates to a composition.

As the enthusiasm for education and growth grows, parents' interest in the growth of their children increases. In particular, interest in growth and height growth is growing, and the interest in the growth of children is high enough that items on the growth plate examination of children in hospitals are essential.

Bone is a tissue composed of calcium and phosphorus, and calcium phosphate complex, collagen fiber, glycoprotein, and proteoglycan are the main constituents, and the organs that make up and support the body. It plays a role in protecting internal organs and supports muscle movement, and also plays a role in constituting and maintaining a shape, but bone marrow can generate blood, an oxygen carrier of the body.

 Osteoblasts, one of the types of bone cells, secrete collagen and alkaline phosphatase to form bones. Alkaline phosphatase does not yet know what function it performs, but it has high bone cell production. It is known to show high levels in the plasma of patients. In addition, osteoclasts are cells that destroy bones, and break down bones to serve to release phosphate and calcium into plasma.

Bone growth is made by the interaction of osteoclasts and osteoblasts. When bone is absorbed by osteoclasts, osteoblasts can be combined again with calcium and phosphorus and bone growth. When osteoblasts have higher activity than osteoclasts, bone growth occurs, and in the opposite case, bone destruction occurs, which can lead to osteoporosis. When osteoblast activity is higher than osteoclasts, growth hormone is secreted at the growth stage, such as children and adolescence.

Calcium and phosphorus, two major constituents of bone, are present in the form of two ions in the complex of bones, but in ionized form in the serum when they move. Depending on the concentration of calcium ions in the blood, calcium resorption is increased or excreted and homeostasis is maintained.

On the other hand, the hormones that affect growth include insulin, thyroid hormone, and sex hormones, among which hormones are secreted from the anterior lobe of the pituitary gland, which grows the body, promotes bone calcification, increases protein synthesis, and immunity. It plays a role in promoting action. Growth hormone is associated with blood sugar because glucose is the energy source available to the brain, the main driver of growth. Excessive growth hormone secretion can lead to high blood sugar levels, which can lead to diabetes.

There is a continuing need for research on growth hormone improvers that help physical health of growing children, prevent metabolic diseases and obesity, and have no side effects.

[Preceding technical literature]

[Patent Documents]

Korea Patent Registration No. 10-1737854

Korea Patent Registration No. 10-0818204

An object of the present invention is to solve the above problems, when administered to a child or adolescents in the growing phase as a medicine, food, etc. in bone growth and growth plate growth of children or adolescents in the growing phase without side effects of metabolic disease or obesity induction Not only to promote bone growth, but also to provide a composition for promoting bone growth, including a fat milk extract that has the effect of maintaining or increasing bone density.

According to one aspect of the invention,

Provided is a pharmaceutical composition for promoting bone growth, including a fat milk extract as an active ingredient.

The fat milk extract may be extracted by water, alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.

The pharmaceutical composition may be prepared from any one of granules, powders, tablets, coated tablets, capsules, solutions, syrups, juices, suspensions, emulsions, drops and injection solutions.

The pharmaceutical composition may be to induce bone growth by enhancing one or more signaling selected from a transforming growth factor beta (TGF-β) receptor and a wnt receptor.

The pharmaceutical composition may be for promoting bone growth of growing children or adolescents.

The pharmaceutical composition may be for promoting bone growth of obese children or adolescents.

According to another aspect of the invention,

Provided is a food composition for promoting bone growth, including a fat milk extract as an active ingredient.

The fat milk extract may be extracted by water, alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.

The food composition may be to induce bone growth by enhancing one or more signaling selected from a transforming growth factor beta (TGF-β) receptor and a wnt receptor.

The food composition may be for promoting bone growth of growing children or adolescents.

The food composition may be for promoting bone growth of obese children or adolescents.

The food composition may be prepared from any one of granules, powders, tablets, coated tablets, capsules, solutions, syrups, juices, suspensions, and emulsions.

According to another aspect of the invention,

It provides a health functional food for promoting bone growth comprising the food composition.

The health functional food may be prepared by adding the fat milk extract to any one food material selected from beverages, teas, spices, gum and confectionery.

According to another aspect of the invention,

Provided is a method of preparing a pharmaceutical composition for promoting bone growth, comprising the step of extracting fat milk with water, alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof to prepare a fat milk extract.

Concentration under reduced pressure may be further performed after the step.

According to another aspect of the invention,

Provided is a method for preparing a food composition for promoting bone growth, comprising the step of extracting fat milk with water, an alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof to prepare a fat milk extract.

Concentration under reduced pressure may be further performed after the step.

According to another aspect of the invention,

Provided is a method for preparing a health functional food for promoting bone growth, wherein the food composition is added to any one food material selected from beverages, teas, spices, gum and confectionery to produce a health functional food.

According to another aspect of the invention,

A novel use of a fat milk extract for the manufacture of a medicament for promoting bone growth is provided.

The medicine for promoting bone growth may be a medicine for preventing or treating a growth disorder.

According to another aspect of the invention,

Provided is a method for treating a growth disorder comprising administering to a patient with a growth disorder a pharmaceutical composition comprising a fat milk extract as an active ingredient.

Bone growth promoting composition comprising the oil extract of the present invention, when administered as a medicine, food, etc. to children or adolescents in the growing phase without the side effects of metabolic diseases or obesity induction, bone length growth, growth plate expansion of children or adolescents in the growing phase In addition to promoting bone growth, bone density is also effective to maintain or increase. It also promotes bone growth and can be used for the prevention or treatment of growth disorders.

1 is a cytotoxicity test results by MTT analysis of osteoblast MG63 cells according to Experimental Example 1.

2 is a result of measuring ALP activity of MG63 cells according to Experimental Example 2.

3 to 5 show the results of mRNA expression analysis of TGF-β signaling-related genes according to Experimental Example 3.

6 and 7 show the results of mRNA expression analysis of wnt signaling-related genes according to Experimental Example 4.

Figure 8 shows the weight measurement results of the white paper after the dietary supply according to Experimental Example 5.

9 is a result of analyzing the length growth effect of tibia (tibia) of the white paper according to Experimental Example 6.

10 is a result of analyzing the growth effect of the femur length of the white paper according to Experimental Example 6.

FIG. 11 is a microscope image of H-E staining of a growth plate in a joint area of a white paper according to Experimental Example 7. FIG.

12 is a graph comparing lengths of growth parts in a growth plate according to Experimental Example 7. FIG.

Figure 13 is a graph comparing the length of the hypertrophy in the growth plate according to Experimental Example 7.

14 is a graph comparing the entire joint length according to Experimental Example 7. FIG.

Figure 15 is the result of measuring the bone density change of the lumbar spine (Lumbar spine) according to Experimental Example 8.

Figure 16 is the result of measuring the bone density change of femur according to Experimental Example 8.

17 is a result of measuring the concentration of ALP in serum according to Experimental Example 9.

18 is a result of measuring the concentration of osteocalcin in the serum according to Experimental Example 9.

As the invention allows for various changes and numerous embodiments, particular embodiments will be illustrated in the drawings and described in detail in the written description. However, this is not intended to limit the present invention to specific embodiments, it should be understood to include all transformations, equivalents, and substitutes included in the spirit and scope of the present invention. In the following description of the present invention, if it is determined that the detailed description of the related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

Fat milk extract of the present invention can be used as an active ingredient of the pharmaceutical composition for promoting bone growth. In addition, the fat milk extract promotes bone growth, and thus may be used for the prevention or treatment of growth disorders. The growth disorder can be normal mutant nephropathy or nephropathy secondary to disease. The normal mutant short stature may be familial short stature, constitutional delay in growth, idiopathic short stature in consultation. In addition, hypothyroidism secondary to disease may be primary growth disorders (endogenous disorders) or secondary growth disorders (exogenous growth disorders), and the primary growth disorders include osteochondral dysplasia, chromosomal abnormalities (Down syndrome or Turner syndrome), short stature due to unreasonable lightweight infants (delayed growth in the uterus), short stature by Freder-Willy syndrome, short stature by Russell-Silver syndrome, and short stature by Nurnan syndrome. Short stature due to systemic diseases, short stature due to growth hormone deficiency, short stature due to hypothyroidism, short stature due to Cushing's syndrome, and psychosocial dwarfism.

In the present invention, the fat milk extract may be extracted using water, an organic solvent or a mixture thereof as an extraction solvent. The kind of organic solvent used at this time and the mixing ratio of water and an organic solvent are not specifically limited.

For example, the organic solvent may be one or more solvents selected from the group consisting of lower alcohols, hexane, acetone, ethyl acetate, chloroform, and diethyl ether. The lower alcohol may be an alcohol having 1 to 6 carbon atoms. For example, methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol, normal-butanol, 1-pentanol, 2-butoxyethanol or ethylene glycol may be used as the lower alcohol. Organic solvents include polar solvents such as acetic acid, dimethyl-formamide (DMFO) and dimethyl sulfoxide (DMSO), acetonitrile, ethyl acetate, methyl acetate, fluoroalkane, pentane, 2,2,4-trimethylpentane, and decane. , Cyclohexane, cyclopentane, diisobutylene, 1-pentene, 1-chlorobutane, 1-chloropentane, o-xylene, diisopropyl ether, 2-chloropropane, toluene, 1-chloropropane, chlorobenzene And nonpolar solvents such as benzene, diethyl ether, diethyl sulfide, chloroform, dichloromethane, 1,2-dichloroethane, anneal, diethylamine, ether, carbon tetrachloride, and THF (Tetrahydrofuran).

As can be seen in the following examples, when the fat milk is extracted with an organic solvent, the fat milk extract is separated into a supernatant and a lower layer solution, the supernatant is an oil fraction of the fat milk extract, the lower layer solution corresponds to a general solvent extract. When the lower layer is separated again by centrifugation or filter paper, it is divided into a liquid phase and a solid residue. The residue is the wax fraction of the fat milk extract. Therefore, the fat milk extract of the present invention is interpreted as a concept including both the oil fraction of the fat milk extract, the liquid fraction of the fat milk extract, and the wax fraction of the fat milk extract. In one embodiment, the fat milk extract may include any one or more selected from the group consisting of an oil fraction of a fat milk extract, a liquid fraction of a fat milk extract, and a wax fraction of a fat milk extract.

In one embodiment, the fat milk extract may be a lower alcohol extract of fat milk, preferably an ethanol extract of fat milk.

As used herein, the term 'extract' also includes fractions that additionally fractionate the extract. That is, the fat milk extract includes not only one obtained by using the aforementioned extraction solvent, but also one obtained by additionally applying a purification process thereto. In addition, fractions obtained by passing the extract or fraction through an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity), etc. The fraction obtained through various purification methods described above is also included in the fat milk extract of the present invention.

In one embodiment, the fat milk extract may be a fraction obtained by re-fractionation of the organic solvent extract of the fat milk with a second organic solvent. Here, the organic solvent extract of the fat milk broadly includes all the oil fraction of the fat milk extract, the liquid fraction of the fat milk extract, the wax fraction of the fat milk extract, and narrowly means the liquid fraction of the fat milk extract. do. Therefore, in one embodiment, the fraction re-fractionated the organic extract of the fat milk with the second organic solvent may mean a fraction that re-fractionated the liquid fraction of the fat milk extract with the second organic solvent. In another embodiment, the fat milk extract may be a fraction obtained by re-fractionation of the lower alcohol extract of fat milk with a second organic solvent. In another embodiment, the fat milk extract may be a fraction of the ethanol extract of the fat milk re-fractionated with hexane. As can be seen in the following examples, the fraction of the fat milk extract containing a large amount of fat-soluble components of such fat milk shows a very good effect.

The term 'extract' as used herein to refer to the fat milk includes not only the crude extract obtained by treating the extraction solvent to the fat milk, but also processed products of the fat milk extract. For example, the fat milk extract may be prepared in powder form by additional processes such as distillation under reduced pressure and freeze drying or spray drying.

In addition, the fat milk extract of the present invention has a meaning broadly also includes a fat milk processed product, such as a fat milk powder, formulated to administer the fat milk itself to an animal. Although the experiment was conducted with a fat milk extract in the present invention, it will be expected to those skilled in the art that the desired effect can be achieved in the same form as the fat milk processed product.

On the other hand, the term 'comprising as an active ingredient' herein means containing an amount sufficient to achieve the efficacy or activity of the fat or milk extract. In one embodiment of the invention, the fat milk extract in the composition of the invention is for example at least 0.001 mg / kg, preferably at least 0.1 mg / kg, more preferably at least 10 mg / kg, even more preferably Is at least 100 mg / kg, even more preferably at least 250 mg / kg, most preferably at least 1 g / kg. Since the fat milk extract has no side effects on the human body even when excessively administered as a natural product, the quantitative upper limit of the fat milk extract contained in the composition of the present invention can be selected and performed by those skilled in the art within an appropriate range.

The pharmaceutical composition of the present invention may be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvant may include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, lubricants. Agents, flavors and the like can be used.

The pharmaceutical composition may be preferably formulated into a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers in addition to the active ingredient described above for administration.

Formulation forms of the pharmaceutical composition may be granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation in the form of tablets or capsules, the active ingredient may be combined with an oral, nontoxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. In addition, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable binders include, but are not limited to, natural and synthetic gums such as starch, gelatin, glucose or beta-lactose, corn sweeteners, acacia, trackercance or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

Acceptable pharmaceutical carriers in compositions formulated in liquid solutions are sterile and physiologically compatible, including saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

Furthermore, the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA can be formulated according to each disease or component, as appropriate in the art.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, or the like, and preferably, oral administration.

Suitable dosages of the pharmaceutical compositions of the present invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to reaction, and usually The skilled practitioner can readily determine and prescribe a dosage effective for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.001-10 g / kg.

The pharmaceutical compositions of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or by incorporating into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or an aqueous medium, or may be in the form of extracts, powders, granules, tablets or capsules, and may further include a dispersant or stabilizer.

The present invention also provides a food composition for promoting bone growth comprising a fat milk extract as an active ingredient.

The food composition according to the present invention may be formulated in the same manner as the pharmaceutical composition, used as a functional food, or added to various foods. Examples of the food to which the composition of the present invention may be added include beverages, alcoholic beverages, confectionary, diet bars, dairy products, meat, chocolates, pizza, ramen noodles, other noodles, gums, ice creams, vitamin complexes, and health supplements. Etc.

The food composition of the present invention may include a fat milk extract or a fat milk powder as an active ingredient, as well as ingredients that are commonly added during food production, and include, for example, proteins, carbohydrates, fats, nutrients, seasonings and flavoring agents. Include. Examples of the above carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And sugars such as conventional sugars such as polysaccharides such as dextrin, cyclodextrin and the like and xylitol, sorbitol, erythritol. As the flavoring agent, natural flavoring agents (tauumatin, stevia extract (for example rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is prepared with a drink and a beverage, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts may be further included in addition to the fat milk extract of the present invention.

The present invention provides a health functional food comprising a food composition for promoting bone growth, comprising the extract as an active ingredient. Health functional food is a food prepared by adding fat milk extract or fat milk powder to food materials such as beverages, teas, spices, chewing gum, confectionery, or by encapsulating, powdering, and suspension. Means to bring, but unlike the general medicine has the advantage that there is no side effect that can occur when taking a long-term use of the drug as a raw material. The health functional food of the present invention thus obtained is very useful because it can be consumed on a daily basis. The addition amount of the fat milk extract or the fat milk powder in such a health functional food cannot be uniformly prescribed | regulated depending on the kind of the health functional food object, but what is necessary is just to add it in the range which does not impair the original taste of food, It is usually in the range of 0.01 to 50% by weight, preferably 0.1 to 20% by weight. In addition, in the case of a health functional food in the form of pills, granules, tablets or capsules, it is usually added in the range of 0.1 to 100% by weight, preferably 0.5 to 80% by weight. In one embodiment, the dietary supplement of the present invention may be in the form of pills, tablets, capsules or beverages.

The present invention also provides the use of a fat milk extract for the manufacture of a medicament or food for promoting bone growth. As described above, the fat milk extract or the fat milk powder may be used for promoting bone growth.

The present invention also provides a method for promoting bone growth, comprising administering an effective amount of a fat milk extract to a mammal.

As used herein, the term "mammal" refers to a mammal that is the subject of treatment, observation or experimentation, preferably human.

As used herein, the term “effective amount” means an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as contemplated by a researcher, veterinarian, doctor or other clinician, Amounts that induce alleviation of the symptoms of the disease or disorder. It will be apparent to those skilled in the art that the effective amount and frequency of administration for the active ingredients of the present invention will vary depending on the desired effect. Therefore, the optimal dosage to be administered can be readily determined by one skilled in the art and includes the type of disease, the severity of the disease, the amount of active ingredients and other ingredients contained in the composition, the type of formulation, and the age, weight, general health of the patient. It may be adjusted according to various factors including the condition, sex and diet, time of administration, route of administration and the rate of secretion of the composition, the duration of treatment, and the drugs used simultaneously.

Compositions comprising a fat or oil extract as an active ingredient in the treatment method of the present invention may be administered in a conventional manner through oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, intraocular or intradermal routes. May be administered.

Advantages and features of the present invention and methods for achieving them will be apparent with reference to the embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in various forms, and only the embodiments are intended to complete the disclosure of the present invention, and the general knowledge in the technical field to which the present invention pertains. It is provided to fully convey the scope of the invention to those skilled in the art, and the present invention is defined only by the scope of the claims.

Hereinafter, preferred examples are provided to aid the understanding of the present invention, but the following examples are merely for exemplifying the present invention, and it will be apparent to those skilled in the art that various changes and modifications can be made within the scope and spirit of the present invention. It is natural that such variations and modifications fall within the scope of the appended claims.

Example: Fat Milk Extract Preparation

Preparation Example 1 Preparation of Fat Milk Extract

After cooling and extracting refrigerated 200 g of 10 times three times with 10 times 70% ethanol three times for 1 hour, the filtrate was concentrated using a vacuum condenser and then lyophilized to prepare a fat milk extract.

Experimental Example: In Vitro Experiment

Experimental Example 1: Cytotoxicity test by MTT analysis of osteoblast MG63 cell

96 well plate 10% FBS, 100 unit / ㎖ penicillin (penicillin) the MG63 cells on, was used as a DMEM culture medium which contains the 100 ㎎ / ㎖ streptomycin (streptomycin), etc., and cultured at 37 ℃, 5% CO ₂ incubator . When the cells were more than 70% filled in each well, the culture solution was removed, and the cells were divided into control and treatment groups, respectively, and cultured under the same conditions. Treatment group was treated with 1 ㎍ / mL and 5 ㎍ / mL fat milk extract to MG63 cells cultured in cultured osteoblasts, and after 1 hour treated with 100 ㎍ / mL palmitate to induce cytotoxicity, After 24 hours, 10 μl of 3- (4,5-dimethythiazol-2yl) -2,5-diphenyl tetrazolium bromide (MTT) solution was added to the control group and the fat treatment group, and then incubated in a CO ₂ incubator for 4 hours. After removing the reaction solution and dissolving formazan crystals formed in the cells by adding 100 μl of dimethyl sulfozide (DMSO), a control group of MG63 cells not treated with fat milk extract, 1 μg / mL fat milk treated group, and 5 μg / mL fat milk Treatment groups were prepared.

By measuring the absorbance at 570 nm wavelength using an ELISA reader, the cell viability of the control group, 1 μg / mL fat milk treatment group, and 5 μg / mL fat milk treatment group was measured and the results are shown in FIG. 1.

The fat milk extract inhibited the cytotoxicity caused by palmitate, inhibited apoptosis and increased cell survival in a concentration-dependent manner, and the high fat milk extract improved cell viability compared to the control group.

Statistical significance between each group was analyzed by one way ANOVA and T-test using prism softward version 7.0 (Graphpad software inc., San Diego, CA, USA).

Experimental Example 2 Measurement of ALP Activity of MG63 Cells

MG63 cells cultured in the same manner as in Experimental Example 1 were prepared, and ALP (alkaline phosphatase) activity was measured in the following manner.

MG63 cells were aliquoted into 96 well plates, and then cultured in a CO ₂ incubator divided into an untreated control group and a fat milk extract treatment group of Preparation Example 1. At this time, in the treatment group, the milk fat extract of Preparation Example 1 was treated at a concentration of 1 µg / ml and 5 µg / ml, respectively. Four days later, supernatants of cultured cells were collected and ALP activity was measured at 405 nm using a kit of Abcam (ab83369). Using the absorbance of the standard, the relationship between the protein concentration and the absorbance was prepared and applied to calculate the protein concentration of the cell solution for each treatment group. The ALP concentration was divided by the protein concentration and applied to the following relationship to calculate the ALP activity, and the results are shown in FIG. 2.

 [expression]

ALP showed the degree of cell destruction, but in cell experiments, osteoblasts were destroyed before new cells were formed, so increasing ALP activity resulted in increased cell growth, and ALP activity was slightly increased in the 5 ㎍ / ml fat-treated group. I did not show a statistically significant difference.

Experimental Example 3: mRNA expression analysis of TGF-β signaling related genes

The results of mRNA expression analysis of TGF-β signaling-related genes in the control group, 1 μg / mL fat milk treatment group, and 5 μg / mL fat milk treatment group of G-63 cells treated under the same conditions as in Experimental Example 1 are shown in FIGS. 5 is shown.

There are several signaling pathways involved in bone cell growth, but when not treated with certain hormones, the growth of bone cell growth increases when transforming growth factor-β (TGF-β and wnt signaling are activated). MRNA expression of the gene was measured.

TGF-β signaling leads to BMP-2-> pSMAD1 / 5/7-> SMAD4-> bone growth, in which DKK1 acts as a mechanism to inhibit TGF-β.

In this study, high concentrations of fat increased the expression of BMP-2, which is likely to improve TGF-β signaling. In TGF-β, fat was enhanced in a concentration-dependent manner, resulting in increased expression of SMAD4, resulting in pSMAD1 / 5/7. The expression of DKK-1, which acts as a negative regulator of TGF-β, was also inhibited in the milk extract. Therefore, the fat milk extract may enhance the signaling of TGF-β receptor, which may improve bone cell division.

Experimental Example 4: mRNA expression analysis of wnt signaling related genes

6 and 7 show the results of mRNA expression analysis of wnt signaling-related genes in the control group, the 1 μg / mL fat milk treatment group, and the 5 μg / mL fat milk treatment group of G-63 cells treated under the same conditions as in Experimental Example 1. Shown in

When LRP involved in wnt signaling was activated, β-catenin was not phosphorylated and moved from the cytoplasm to the nucleus to increase the expression of wnt target genes, thereby increasing wnt signaling. The fat milk extract is thought to increase cell growth through TGF-β and wnt signaling in osteoblasts.

Example: Fat Milk Extract Administration

Example 1

Four-week-old male rats were fed orally 100 mg / kg body weight (0.015% diet) for 4 weeks orally with a fat milk extract prepared according to Preparation Example 1 together with a diet adjusted to only 43 calories percent fat in AIN-93G feed. .

Example 2

The diet was provided in the same manner as in Example 1 except that the milk extract did not provide 100 mg / kg body weight (0.015% diet) and 300 mg / kg body weight (0.045% diet).

Negative control

The diet was provided in the same manner as in Example 1, except that the fat milk extract was not provided.

Positive control

The diet was provided in the same manner as in Example 1, except that subcutaneous injection of 20 μg / kg body weight (Eutropin-L Life Science) was used instead of the fat milk extract.

Experimental Example: In Vivo Test

Experimental Example 5: Weight measurement after diet

FIG. 8 is a graph comparing body weights of 4 male rats after 4 weeks of feeding AIN-93G feed adjusted to only 43 calories of fat content.

According to this, there was no statistically significant difference between the control group and the fat milk extract administration group after feeding the diet for 4 weeks.

Experimental Example 6: Analysis of Bone Growth Effect

The growth of the tibia and femur of the white paper was observed and the results are shown in FIGS. 9 and 10 below.

9, after feeding for 4 weeks, the length of tibia was statistically significantly increased in the positive control group (36.704) compared to the negative control group (35.966 mm), and Example 2 was negative in 37.592 mm. It was 104.52% longer than the control and showed a larger value than the positive control.

According to FIG. 10, the femur length was not significantly different from the negative control group in the negative control group after feeding the diet for 4 weeks, but in Example 2, it was statistically significantly longer than the negative control group. Could.

Experimental Example 7: H-E staining of growth plate

HE microscopic images of growth plates in the joint area of the white paper are shown in FIG. 11, and graphs comparing the lengths of the proliferation part and the hypertrophy part in the growth plate are shown in FIGS. 12 and 13. The graph is shown in FIG.

In other cases, we examined whether the nucleus increased constantly. The negative control group showed a small growth plate area and irregular cells. The positive control group showed a wide growth plate area and the cell nuclei were regularly arranged. Could see.

In Examples 1 and 2, the growth plate region was widened in a concentration-dependent manner and the cells were regularly arranged as compared to the negative control group.

Experimental Example 8: Observation of Bone Mineral Density

After 4 weeks, bone mineral density (BMD) of the lumbar spine and bone density of the femur were measured and shown in FIGS. 15 and 16, respectively.

For 4 weeks, the increase in bone mineral density (BMD) of lumbar spine was greater in Example 2 than in the negative control group, and the increase in BMD of femur was also higher in Example 2 than in the negative control group. The positive control group also increased bone density of the femur compared to the negative control group.

Experimental Example 9: ALP Concentration in Serum

The dissection of the white paper and the results of measuring the concentration of ALP (alkaline phosphatase) in serum is shown in Figure 17, the concentration of osteocalcin in serum is shown in Figure 18.

According to this, the ALP concentration indicating the degree of bone degradation was lower in Example 2 than in the negative control group, and showed a similar value to the positive control group.

In addition, the concentration of osteocalcin in serum, which is an indicator of the degree of bone synthesis, was higher in Example 2 than in the negative control group and was not statistically different from the positive control group.

As a result, the length of femur and tibia is longer in Example 2 than that of the negative control and positively correlated with serum osteocalcin concentration.

As described above, embodiments of the present invention have been described, but those skilled in the art may add, change, delete, or add elements within the scope not departing from the spirit of the present invention described in the claims. The present invention may be modified and changed in various ways, etc., which will also be included within the scope of the present invention.

Claims (17)

1. A pharmaceutical composition for promoting bone growth, comprising a fat extract and a pharmaceutically acceptable carrier.
2. The method of claim 1,

   The fat milk extract is a bone growth promoting pharmaceutical composition, characterized in that extracted with water, alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.
3. The method of claim 1,

   The bone growth promoting composition is a bone growth promoting pharmaceutical composition, characterized in that to induce bone growth by enhancing one or more signaling selected from the TGF-β (Transforming growth factor beta) receptor and wnt receptor.
4. The method of claim 1,

   The bone growth promoting composition is bone growth promoting pharmaceutical composition, characterized in that for promoting bone growth of children or adolescents.
5. The method of claim 4, wherein

   The bone growth promoting composition is a bone growth promoting pharmaceutical composition, characterized in that for promoting bone growth of obese growth children or adolescents.
6. Bone growth promoting food composition comprising a fat milk extract as an active ingredient.
7. The method of claim 6,

   The fat milk extract is a food composition for promoting bone growth, characterized in that extracted with water, alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.
8. The method of claim 6,

   The bone growth promoting composition is a bone growth promoting food composition, characterized in that to induce bone growth by improving the signal transmission of at least one selected from TGF-β (Transforming growth factor beta) receptor and wnt receptor.
9. The method of claim 6,

   The bone growth promoting composition is bone growth promoting food composition, characterized in that for promoting bone growth of children or adolescents.
10. The method of claim 6,

    The bone growth promoting composition is a bone growth promoting food composition, characterized in that for promoting bone growth of obese children or adolescents.
11. A method of preparing a pharmaceutical composition for promoting bone growth, comprising the step of extracting fat milk with water, alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof to prepare a fat milk extract.
12. The method of claim 11,

    Method of producing a pharmaceutical composition for promoting bone growth, characterized in that further performing the step of concentration under reduced pressure after the step.
13. A method of producing a food composition for promoting bone growth, comprising the step of extracting fat milk with water, an alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof to prepare a fat milk extract.
14. The method of claim 13,

    Method of producing a food composition for promoting bone growth, characterized in that for performing the further step of concentration under reduced pressure after the step.
15. Use of fat milk extract for the manufacture of a medicament for promoting bone growth.
16. The method of claim 15,

    The bone growth promoting medicament is a use of a fat milk extract, characterized in that the medicament for the prevention or treatment of growth disorders.
17. A method of treating growth disorders comprising administering to a patient with growth disorders a pharmaceutical composition comprising fat extract as an active ingredient.

Images