KR101109638B1 - Composition for Improving Osteoporosis - Google Patents

Abstract

The present invention discloses osteoporosis improver compositions. Specifically, the present invention discloses an osteoporosis improving composition comprising the horse bone extract as an active ingredient.

Horse bones, osteoporosis

Description

Composition for improving osteoporosis {Composition for Improving Osteoporosis}

The present invention relates to an osteoporosis improving composition, and more particularly to an osteoporosis improving composition comprising a horse bone extract as an active ingredient.

Contrary to what we generally know, humans continue to synthesize and degrade bones not only during their growth but also during their lives (Mundy et al., 1993). The degradation of bone continues during the growing season, but the growth continues because the synthesis occurs more actively than the decomposition of bone. However, in the elderly, where the environmental imbalance, hormonal imbalance caused by physical or mental changes of the body, or the decomposition is more active than making bones, bone breaks easily occur even with a small impact.

Osteoporosis, which often occurs in postmenopausal women, is a disease caused by the formation of spaces between bones, making it difficult to make bones, and more active in breaking bones, due to hormone imbalances in the body. to be. In addition, osteopetrosis is a disease in which bone breakdown does not occur well due to calcification of the space in the bone, the development of the bone does not occur, or the arms and legs are short, and the formation of bone marrow, which is the basis of all immune cells, does not occur. This also happens. This osteoporosis can also break easily like bone with osteoporosis because the bone is too hard. In Korea, however, osteoporosis occurs more frequently than osteoporosis, and osteoporosis is emerging as a post-menopausal problem worldwide (Teitelbaum et al., 1996).

Bone is a specialized tissue that combines a hardened calcified surface with an internal cellular component called the bone marrow. The combination of these two physiologically different structures is due to a lifelong process of bone remodeling, which causes osteoclasts in the bone marrow to reach the bone's surface due to hormones or physical stimuli applied to the bone. It is explained by bone resorption that collects and destroys bone and destroys bone matrix by osteoblasts gathered at the site where absorption occurs (Park, 2000).

Thus, bone resorption inhibitors or bone synthesis promoters are currently being used for the treatment of osteoporosis. Estrogen, calcitonin, bisphosphonate, and the like are bone absorption inhibitors, and fluorine, parathyroid hormone, vitamin D metabolites, and calcium are bone synthesis promoters.

The present invention is also completed by confirming the fact that the horse bone powder extract has a bone absorption inhibitory activity and bone formation promoting activity.

An object of the present invention to provide an osteoporosis improving agent composition using a horse bone powder extract having a bone resorption inhibitory activity and bone formation promoting activity.

Other objects or specific aspects of the present invention will be presented below.

The present inventors prepared the hydrothermal extract, ethanol aqueous solution (ethanol content 40% to 90%) extract and ethanol (ethanol 100%) extract of horse bone powder as shown in the following Examples and Experimental Examples and osteoblast cells of the extract The proliferation promoting activity and inhibitory activity of osteoclasts were examined. The hydrothermal extract showed little activity, but the ethanol aqueous extract and ethanol extract showed osteoblast proliferation promoting activity and osteoclast differentiation inhibitory activity. As described above, osteoblasts are cells responsible for bone synthesis, and osteoclasts are cells responsible for bone resorption.

The present inventors measured the bone density from the femur to the toe bone through animal experiments based on the results of these cell experiments, and as shown in the following experimental example, the measurement of the bone density was similar to the cell experiments. Aqueous extract and ethanol extract have been shown to improve bone density.

In particular, the ethanol aqueous extract and the extract with ethanol content of 60% to 80% in the ethanol extract was high activity, the extract with the ethanol content of 70% to 80% was the highest activity.

In view of the above, the present invention can be understood as an osteoporosis improving agent composition comprising an ethanol aqueous solution extract or ethanol extract of horse bone as an active ingredient.

The ethanol aqueous solution is preferably 60% to 80% ethanol aqueous solution, and particularly preferably 70% to 80% ethanol aqueous solution of ethanol. Where% means volume percentage (v / v).

In the present specification, the term "active ingredient" means a component that can exhibit activity alone or in combination with a carrier which is not active in itself.

In the present specification, the term "improvement" is meant to include the prevention, treatment and alleviation of pathological symptoms.

Osteoporosis improver composition of the present invention may include an aqueous ethanol extract or ethanol extract of the horse bone as an active ingredient in any amount (effective amount) as long as it can exhibit an osteoporosis improving activity according to the use, formulation, formulation purposes, etc. An effective amount of phosphorus will be determined within the range of 0.001% to 15% by weight, based on the total weight of the composition. As used herein, an "effective amount" refers to an amount of an effective ingredient capable of inducing, preventing, treating, or alleviating osteoporosis in a mammal or a human. Such effective amounts can be determined experimentally within the range of ordinary skill in the art.

The composition of the present invention can be used as a pharmaceutical composition in a specific embodiment.

The pharmaceutical composition of the present invention includes oral formulations (tablets, suspensions, granules, emulsions, capsules, syrups, etc.), including pharmaceutically acceptable carriers, excipients, etc. in addition to the ethanol extract or ethanol extract of horse bone, which is an active ingredient, Parenteral formulations (aqueous or oily suspensions for sterile injection), topical formulations (solutions, creams, ointments, gels, lotions, patches) and the like.

As used herein, "pharmaceutically acceptable" means that the subject of application (prescription) does not have more toxicity (adequately low toxicity) to which the subject of application (prescription) is adaptable without inhibiting the activity of the active ingredient.

Examples of pharmaceutically acceptable carriers include lactose, glucose, sucrose, starch (such as corn starch, potato starch, etc.), cellulose, derivatives thereof (such as sodium carboxymethyl cellulose, ethylcellulose, etc.) malt, gelatin, talc, solids Lubricants (e.g. stearic acid, magnesium stearate, etc.), calcium sulfate, vegetable oils (e.g. peanut oil, cottonseed oil, sesame oil, olive oil, etc.), polyols (e.g. propylene glycol, glycerin, etc.), alginic acid, emulsifiers (e.g. TWEENS), wetting agents (e.g. Sodium lauryl sulfate), colorants, flavoring agents, tableting agents, stabilizers, antioxidants, preservatives, water, saline, phosphate buffer solutions and the like. The carrier may be selected from one or more of suitable pharmaceutical formulations according to the formulation of the pharmaceutical composition of the present invention.

Excipients may be selected and used according to the formulation of the pharmaceutical composition of the present invention, for example, when the pharmaceutical composition of the present invention is prepared with an aqueous suspending agent, suitable excipients are sodium carboxymethyl cellulose, methyl cellulose, hydroxypropylmethyl Suspending agents and dispersing agents, such as cellulose, sodium alginate, and polyvinylpyrrolidone, etc. are mentioned. Suitable excipients when prepared from injection solutions include Ringer's solution, isotonic sodium chloride, and the like.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and in some cases, can be administered topically.

The daily dose of the pharmaceutical composition of the present invention is usually 0.001 to 150 mg / kg body weight, and may be administered once or several times. However, since the dosage of the pharmaceutical composition of the present invention is determined in view of various related factors such as the route of administration, the age, sex, weight of the patient, the severity of the patient and the like, the dosage may limit the scope of the present invention in any aspect. It should not be understood as.

The composition of this invention can be grasped | ascertained as a food composition in another specific aspect.

The food composition of the present invention may be prepared from gums, vitamin complexes, dietary supplements, special nutritional supplements, functional drinks, and the like.

The food composition of the present invention includes, in addition to the horse bone extract as an active ingredient, sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose, maltose, fruits such as apples, lemons, citrus fruits, green tea leaves, round stalks, Flavoring agents obtained from teas such as jujube leaves, bioactive components such as catechin, retinol, ascorbic acid and tocopherol, minerals such as calcium, magnesium, chromium, cobalt, copper and fluoride may also be added.

In addition, herbal medicines may be added to enhance flavor and palatability and to add other functionalities (such as prevention of arthritis). Herbal medicines that may be added include tofu, fast, antler, safflower, earthenware, succinate, tortoise, cornus, goji berry, Licorice, Angelica, etc. can be illustrated.

In addition, the food composition of the present invention in order to increase and enhance the effect of improving osteoporosis, the root extract (Korean Patent No. 348148), Kang Jinhyang Extract (Korean Patent No. 457217), Haphwanpi extract ( Republic of Korea Patent No. 380867), mountain triceps extract (Korean Patent No. 380866), lump extract (Korean Patent No. 380865), ginseng extract (Korean Patent No. 380864) and the like may further be included.

As described above, the present invention can provide an osteoporosis improving composition comprising a horse bone extract, in particular, an aqueous ethanol extract of a horse bone or an ethanol extract of a horse bone as an active ingredient. Osteoporosis improver compositions of the present invention may be formulated into pharmaceutical compositions or food compositions.

Hereinafter, the present invention will be described with reference to Examples and Experimental Examples. However, the scope of the present invention is not limited to these examples.

<Examples> Preparation of Horse Bone Powder Extract

Example 1 Preparation Example 1 of Horse Bone Powder Extract

The horse bone used in the experiment was Jeju horse, which was purchased through a horse meat distributor in Jeju. Horse bone crushing was milled to about 5mm through a processing mill to obtain a horse bone powder 12kg, which was first dried in a hot air dryer and used in this experiment while frozen storage.

5 kg of horse bone powder was placed in 50 L of water and extracted by boiling for 24 hours at a temperature of 105 ° C. using a hot water extractor (HD-A80), and then lyophilized and powdered and stored at -20 ° C. until used in experiments.

Example 2 Preparation Example 2 of Horse Bone Powder Extract

1kg of horse bone powder of <Example 1> was immersed in 40% ethanol aqueous solution, shaken at room temperature for 24 hours, filtered with Whatman No. 2, and the filtrate was decompressed and concentrated before experimenting. Store at -20 ° C.

Example 3 Preparation Example 3 of Horse Bone Powder Extract

To prepare a horse bone extract in the same manner as in <Example 2>, the extraction solvent was used instead of 40% aqueous ethanol solution of 50% ethanol.

Example 4 Preparation Example 4 of Horse Bone Extract

The horse bone extract was prepared in the same manner as in <Example 2>, except that the extraction solvent was used for the aqueous solution of 60% ethanol instead of 40% ethanol.

Example 5 Preparation Example 5 of Horse Bone Extract

The horse bone extract was prepared in the same manner as in <Example 2>, but 70% ethanol aqueous solution was used as the extraction solvent instead of 40% ethanol aqueous solution.

Example 6 Preparation Example 6 of Horse Bone Extract

The horse bone extract was prepared in the same manner as in <Example 2>, but 80% ethanol aqueous solution was used instead of 40% ethanol aqueous extraction solvent.

Example 7 Preparation Example 7 of Horse Bone Extract

To prepare a horse bone extract in the same manner as in <Example 2>, 90% ethanol aqueous solution was used as the extraction solvent instead of 40% ethanol aqueous solution.

Example 8 Preparation Example 8 of Horse Bone Extract

To prepare a horse bone extract in the same manner as in <Example 2>, 100% ethanol was used as the extraction solvent instead of 40% ethanol aqueous solution.

Experimental Example Osteoporosis Improvement Activity Test of Horse Bone Extract

Experimental Example 1 Search for Growth Rate of Osteoblasts

MG-63 cells and SaOS-2 cells, which are human osteoblast cell lines, were obtained from Korean Cell Line Bank (KCLB) and contained DMEM medium containing 100 units / ml of penicillin-streptomycin and 10% fetal bovine serum (FBS). Was cultured at 37 ℃, 5% CO ₂ incubator, MG-63 cells and SaOS-2 cells were passaged once every 4 days.

The proliferation rate of osteoblasts of each sample was determined by MTT assay (Carmichael et al., 1987). Osteoblasts (1.0 × 10 ⁵ / mL) were placed in each well of a 96 well plate and samples were added at a concentration of 100 μg / mL. After incubating for 4 days, 100 μg of 3- (4,5-dimethylthiazol) -2,5-diphenyltetrazolium bromide (MTT; Sigma, St. Louis, Mo., USA) was added and further incubated for 4 hours. The plate was centrifuged at 1,000 rpm for 10 minutes, and the medium was carefully removed. Then, 150 μL of dimethylsulfoxide (DMSO; Sigma, St. Louis, MO, USA) was added to dissolve the formazan precipitate produced by the reduction of MTT. Absorbance was measured at 540 nm using Bio-TEK instrumenis. Inc., Winooski Vermont, WI, USA). The average absorbance value for each sample group was obtained, and the cell proliferation rate was measured by comparing with the absorbance value of the control group.

The results are shown in Table 1 below.

[Table 1]

Osteoblast proliferation

Example 2 Measurement of Inhibitory Activity of Osteoclasts

Example 2-1 Cell Culture

RAW 264.7 cells were obtained from Korean Cell Line Bank (KCLB) and cultured in 37 ° C, 5% CO2 incubator using DMEM medium containing 100 units / mL of penicillin-streptomycin and 10% fetal bovine serum (FBS). Subcultures were performed once a day.

Example 2-2 Measurement of Differentiation Inhibitory Activity into Osteoclasts

RAW264.7 cells were adjusted to 1.5 × 10 ⁵ cells / mL using DMEM medium containing 10% FBS, inoculated into 48 well plates, and treated with RANKL 100ng / mL to induce differentiation into osteoclasts. . After 1 day, the samples of each of the above examples were treated at a concentration of 100 µg / ml and cultured for 3 days. After taking a certain amount of medium, the supernatant was centrifuged and the amount of TRAP of the osteoclast indicator factor was quantified using a TRAP assay kit, and is shown in <%> in terms of the control group. In the results of Figure 1, the ethanol extract is generally high in osteoclast differentiation inhibitory activity, in particular 60% ethanol extract, 70% ethanol extract and 80% ethanol extract is high activity.

Example 2-3 Cytotoxicity Measurement

RAW264.7 cells were adjusted to 1.5 × 10 ⁵ cells / mL, inoculated in 48 well plates, incubated for 24 hours, and then the samples of each Example were incubated for 3 days after treatment at a concentration of 100 ng / mL. 100 μg of 3- (4,5-dimehtylthiazol) -2,5-diphenyltetrazolium bromide (MTT, Sigma) was added thereto and further incubated for 4 hours. The plate was centrifuged at 1000 rpm for 10 minutes and the medium was carefully removed. Then, 150 μl of dimethylsulfoxide (DMSO, Sigma) was added to dissolve the formazan precipitate produced by the reduction of MTT. The absorbance was measured at 540 nm using a microplate reader. Measured. The average absorbance value for each sample group was obtained, and the degree of growth inhibition was investigated by comparing with the absorbance values of the control group.

The results are shown together in FIG. 1. Overall, each extract of the example did not show cytotoxicity. This shows that the osteoclast differentiation inhibitory activity of the extract of each of the above examples was not due to cytotoxicity.

Example 3 Measurement of Bone Mineral Density

Intradermal injections of complete Freund's adjuvant ( Mycobacterium tuberculosis , Sigma, USA) at a dose of 0.5 ml / rat in SD rats induced arthritis and the day of arthritis was day 0. Arthritis is known to cause osteoporosis when chronically advanced. Thereafter, the extract of each example was administered for 1000 mg / kg / day for 28 days, and then bone density from the femur to the toe was measured. The measurement of the bone density was measured by the dual energy X-ray absorption method (DEXA). BMC (Bone mineral contents) was measured by pDEXA Saber (Norland medical Systems, Fort Atkinson, WI, USA) and analyzed by Sabre Research software (version 3.6; Norland). Bone mineral density (BMD) was calculated by dividing BMC by the area of bone measured.

The results are shown in FIG. 2. The results of FIG. 2 also show that the ethanol extract, particularly 60% to 80% ethanol extract, has high activity as shown in Table 1 and FIG. 1.

Statistical analysis

The experimental results were analyzed using a SAS program (statistical analysis system) and expressed as mean and standard deviation. Duncan's multiple range test was performed at the significance level of p <0.05 to analyze the statistical difference between the groups.

1 is a graph showing the osteoclast differentiation inhibitory activity of the horse bone extract. In FIG. 1, Control is a non-treated group, a negative control and RANKL is a positive control.

Figure 2 is a graph showing the effect of horse bone extract on bone density. In FIG. 2, N / C is a negative control group not induced arthritis, and A / C is a positive control group induced arthritis. N / C and A / C were administered Saline instead of horse bone extract. ^*** P <0.001 is statistical significance for N / C, ⁺ P <0.05 and ⁺⁺ P <0.01 are statistical significance for A / C.

Claims (6)

delete delete delete delete delete Osteoporosis improver food composition comprising the horse bone extract obtained by immersing the horse bone powder in 70% ethanol aqueous solution and extracted with stirring for 24 hours.

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