KR101152753B1 - Pharmaceutical composition for preventing and treating of metabolic bone disease comprising the harpagophytum - Google Patents

Abstract

PURPOSE: A pharmaceutical composition containing Harpagophytum procumbens DC. extract is provided to suppress osteoclast differentiation and to treat bone metabolic diseases. CONSTITUTION: A pharmaceutical composition for preventing and treating bone metabolic diseases contains 100 weight parts of Harpagophytum procumbens DC. extract, 10-300 weight parts of Drynaria fortunei (Kze.) J. sm., 50-500 weight parts of Equus asinus L. extract, 50-500 weight parts of Poria cocos extract, 10-300 weight parts of Rehmannia glutinosa LIBOSCH., and 10-300 weight parts of ginseng extract. The Harpagophytum procumbens DC. extract is prepared using water, low carbon number alcohol, or mixture solvent thereof.

Description

PHARMACEUTICAL COMPOSITION FOR PREVENTING AND TREATING OF METABOLIC BONE DISEASE COMPRISING THE HARPAGOPHYTUM}

The present invention relates to a pharmaceutical composition for the prevention and treatment of bone metabolic diseases, including Harpagopitum muscle extract as an active ingredient, more specifically osteoblast proliferation, differentiation promoting activity, and osteoclast differentiation The present invention relates to a pharmaceutical composition for preventing and treating bone metabolic diseases, or a food or health functional composition for promoting bone strengthening or growth development, comprising an extract of harpagopitum muscle having inhibitory activity as an active ingredient.

Bone tissue is a dynamic tissue in which absorption and formation are constantly occurring to maintain bone mass and skeletal homeostasis. This bone remodeling involves two specially functioning cells. Osteoclasts absorb bone, while osteoblasts play a role in synthesizing and filling bone matrix. Thus, bone mass will depend on the relative function of these cells. There is always a balance between bone resorption and bone formation in normal adults. Bone remodeling occurs at regular intervals and occurs locally in narrow areas to maintain the structure of the skeletal system, which is regulated by the complex interactions between systemic hormones and local factors.

The imbalance between osteoblast and osteoclast activity is due to skeletal abnormalities caused by osteoporosis or osteosclerosis. There are many theories and arguments as to whether this metabolic imbalance is primarily caused by osteoblast degeneration or increased bone resorption, that is, by osteoclast activity. Deficiency, lack of exercise, lack of calcium, genetic factors, drug use, hormone effects

Osteoporosis, which has emerged as a major problem in the elderly society among bone metabolic diseases, is a disease that can easily cause fractures even with a slight impact due to a decrease in the amount of bone in the unit volume without a great change in the chemical composition of the bone. The elderly, especially postmenopausal women, are known to have the highest incidence due to lack of estrogen (Jilka RL. Cytokines, bone remodeling and estrogen deficiency. Bone 23: 75-81 (1998)). However, even in men, bone mass tends to decrease with age (Stein GS, Lian JB, van Wijnen AJ, Montechino M. Transcriptional control of osteoblast growth and differentiation.Physiol Rew. 76: 593-629 (1996)). In 20 years, the fracture rate of elementary and middle school students has more than doubled. In terms of the prevention or treatment of bone diseases due to bone metabolic imbalances, it is very important to increase bone mass from a young age, and thus maintaining bone health is an important issue regardless of gender or age.

Most of the drugs currently used for the prevention and treatment of osteoporosis do not completely restore the progression of bone loss because they act to inhibit bone resorption, and thus, the ultimate goal of osteoporosis cannot be prevented completely. In order to prevent and treat osteoporosis, research on increasing bone formation has recently been attracting attention, and a scientific approach to the effects on bone tissue regeneration is required.

Harpagopitum muscle is nicknamed 'Hrpagophytum procumbens DC' inhabiting the Kalahari desert in Africa and is a Pelaliaceae family that uses rhizome as a folk medicine in Africa and Europe. The components of the Harpagopitum muscle include harpagide, harpagoside, procumbide, 8-O- (para-cumanoyl) -harpazide [8-O- (p -coumaroyl) -harpagide], 6'-O- (para-coumanoyl) -procumbid [6'-O- (p-coumaroyl) -procumbide], procumboside, are known, but Pharmacological action is unknown.

The present invention provides a pharmaceutical composition comprising a Harpagopitum muscle extract as an active ingredient that promotes proliferation and differentiation of osteoblasts and osteocalcification, and at the same time inhibits osteoclast differentiation to effectively prevent or treat bone metabolic diseases. The purpose is to provide.

It is another object of the present invention to provide a health functional food useful for strengthening and growing the bone during the growth phase containing the Harpagopitum muscle extract.

The present invention provides a pharmaceutical composition for the prevention and treatment of bone metabolic diseases comprising harpagopitum muscle extract as an active ingredient to solve the above problems.

In the present invention, the pharmaceutical composition for preventing and treating bone metabolic diseases is characterized in that it has osteoblast proliferation, differentiation promoting activity, and osteoclast differentiation inhibitory activity.

In the present invention, the Harpagopitum root extract is characterized in that it is obtained by extracting the powder of Harpagopitum root in water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof.

The pharmaceutical composition for the prevention and treatment of the bone metabolic disease of the present invention is 10 to 300 parts by weight of bone chain beam, 100 to 500 parts by weight of glue, 50 to 500 parts by weight, Fuling 50 to 500 parts by weight, 100% by weight of the harpagopitum muscle extract It is characterized by comprising 10 parts by weight to 300 parts by weight and ginseng extract.

Drynaria fortunei (Kze. J. sm.) Is a dried perennial herb perennial herb, or rhizome of bone clavicle. Its ingredients are warm, bitter, non-toxic, and have bleeding and hemostatic effects. In particular, it is known as an important drug for treating chronic neuralgia. The main ingredients are hesperidin, naringin, starch and glucose. have.

In general, Fukryeong is a fungal parasitic nucleus around pine roots 5 to 6 years after ablation of pine trees, including pale white baekbokyeong, red brown bokbokyeong, and pine root. In oriental medicine, Fuling extracts are classified as sedatives and diuretics. In addition, Bokryeong extract is used as one of the important components of herbal preparations for energy recovery. Studies and experiments related to the pharmaceutical efficacy of Bokryong extract in recent years, it has been found that Bokryong extract is excellent in suppressing tumors and improves the immunity of people suffering from chronic diseases.

Rehmannia glutinosa LIBOSCH. Is a herb that belongs to Hyunsam and its main ingredient is Catalpol, a kind of Iridoid glycoside, and has physiological activities such as diuresis and hypogonadism. In particular, the myopia has a tonic tonic effect and is widely used in various herbal prescriptions and herbal preparations for the purpose of treating anemia, weakness and diabetes.

Ginseng is a perennial herb belonging to the Ogapiaceae, and saponin-based derivative compounds having various physiological activities have main effects, and ginsenoside content is reported to be an indicator of quality. In Chinese medicine, gunpowder powder, suit

It is used in prescriptions such as anti-diarrheal drugs and analgesic drugs. The pharmacological action includes hypoglycemic action, anticancer effect, serum protein synthesis promotion, antibody production promotion action, central excitability action, and anti-fatigue action.

Each extract of the extract is a drug that has been used as a herbal medicine for a long time, the extracts of the present invention extracted therefrom also have no problems such as toxicity and side effects. Each component may be mixed and then extracted at once and a method of mixing each extract may be used.

In the present invention, the bone metabolic disease includes osteoporosis, Paget's disease, periodontal disease, bone metastasis cancer or rheumatoid arthritis.

The pharmaceutical composition for the prevention and treatment of bone metabolic diseases comprising the harpagopitum muscle extract of the present invention as an active ingredient may further comprise appropriate carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Pharmaceutical compositions for the prevention and treatment of bone metabolic diseases comprising the Harpagopitum muscle extract of the present invention as an active ingredient, respectively, powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. Carriers, excipients and diluents which may be formulated in the form of oral dosage forms, external preparations, suppositories, and sterile injectable solutions, and which may be included in the composition comprising the compound, 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 such solid preparations may contain at least one excipient such as starch, calcium carbonate, sucrose, or the like. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and 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.

The preferred dosage of the pharmaceutical composition for preventing and treating bone metabolic diseases, including the Harpagopitum muscle extract of the present invention as an active ingredient, depends on the condition and body weight of the patient, the extent of the disease, the form of the drug, the route and duration of administration. It may be appropriately selected by those skilled in the art. However, for the desired effect, the pharmaceutical composition for the prevention and treatment of bone metabolic diseases comprising the Harpagopitum muscle extract of the present invention as an active ingredient is 0.01 mg / kg to 10 g / kg, preferably 1 mg / day Administration at kg to 1 g / kg is preferred. The administration may be carried out once a day or divided into several doses. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.

The composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, etc. by various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

The present invention also provides a health functional food for the prevention and improvement of bone metabolic diseases comprising the Harpagopitum muscle extract as an active ingredient.

In the present invention, the health functional food for preventing and improving the bone metabolic disease is characterized by having osteoblast proliferation, differentiation promoting activity, and osteoclast differentiation inhibitory activity.

The present invention provides a health functional food comprising the Harpagopitum muscle extract of the present invention as an active ingredient. Harpagopitum root extract of the present invention is a compound that can be used with confidence even for long-term administration for the purpose of prevention because there is little toxicity and side effects.

Health functional food containing the Harpago pitumugeun extract of the present invention as an active ingredient can be used in a variety of drugs, foods and beverages for the prevention and improvement of bone metabolic diseases. Foods to which the alpha-tocopherol succinic acid of the present invention can be added include, for example, various foods, beverages, gums, teas, vitamin complexes, dietary supplements, and the like, which are powders, granules, tablets, capsules, or beverages. Can be used as

Harpagopitum muscle extract of the present invention can be added to food or beverage for the purpose of preventing and improving bone metabolic diseases. At this time, the amount of the compound in the food or beverage is generally added to the health food composition of the present invention to 0.01 to 15% by weight of the total food weight, the health beverage composition is 0.02 to 10 g, preferably based on 100 ml Can be added in a ratio of 0.3 to 1 g.

The health beverage composition of the present invention, in addition to containing the compound as an essential ingredient in the indicated proportions, has no particular limitation on the liquid component 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 disaccharides such as glucose and fructose such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like Sugar, 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 said natural carbohydrate 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 composition 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 compositions of the present invention may contain flesh for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

The pharmaceutical composition for preventing and treating bone metabolic diseases comprising the extract of harpagopitum muscle according to the present invention as an active ingredient promotes the proliferation and differentiation of osteoblasts and osteocalcification, and promotes the differentiation of osteoclasts. It shows an inhibitory effect and shows an effect effective for improving bone metabolic diseases including osteoporosis.

Figure 1 shows the results of measuring the cell viability by performing the MTT assay when the composition containing the Harpagopitum root extract of Example 1 and the Harpagopitum root extract of Example 2 of the present invention.
2 and 3 show the results of measuring the ALP activity when the composition containing the Harpagopitum root extract of Example 1 and the Harpagopitum root extract of Example 2 of the present invention.
4 and 5 show the results of measuring the degree of calcification by AR staining when the composition containing the Harpagopitum root extract of Example 1 of the present invention and the Harpagopitum root extract of Example 2 was treated.
6 and 7 show the results of measuring the cytotoxicity to osteoclasts when the composition containing the Harpagopitum root extract of Example 1 of the present invention and the Harpagopitum root extract of Example 2 was treated.
8 and 9 show the results of measuring the degree of inhibition of osteoclast activity by TRACP staining when the composition containing the Harpagopitum root extract of Example 1 of the present invention and the Harpagopitum root extract of Example 2 were treated. Indicates.
Figure 10 shows the change in body weight of the rats to which the composition containing the Harpagopitum root extract of Example 1 and the Harpagopitum muscle extract of Example 2 of the present invention.
11 and 12 are structures of the dimensional fine spongy bone and cortical bone of the femur isolated from the rat administered the composition containing the Harpagopitum root extract of Example 1 and the Harpagopitum muscle extract of Example 2 and The indicators of the microstructures are measured.
13 and 14 show the results of measurement of the activity of bone biomarker in blood of rats to which the composition containing the Harpagopitum muscle extract of Example 1 of the present invention and the Harpagopitum muscle extract of Example 2 was administered .

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the present invention is not limited by the following examples.

< Example  1> Preparation of Harpagopitum Muscle Extract

8,70% ethanol was added to 1,205 g of harpagopitum muscle powder, and extracted four times for 6 hours to obtain x 598 g of harpagopitum muscle. The Harpagopitum root extract was suspended in 1 L of water, extracted three times with 1 liter of a butanol / hexane mixed solvent, and concentrated to obtain 12 g of Harpagopitum root extract.

< Example  2> Preparation of a pharmaceutical composition for the prevention and treatment of osteoporosis comprising the Harpago pitumeun extract as an active ingredient

To prevent and treat osteoporosis, which contains Harpagopitum root extract as an active ingredient by adding 10g, Goljubo 5g, 5g, Fuling 5g, Ginseng extract 2g and 5g ginseng extract to 10g of the Harpagopitum root extract prepared in Example 1 A pharmaceutical composition was prepared.

< Experimental Example  1> Bone  Activation efficacy analysis of cells

< Experimental Example  1-1> Bone  Cell culture

As osteoblasts, MC3T3-E1 was purchased from ACTC and used. The MC3TC-E1 cells contained 37%, 5% in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U / mL penicillin and 100 μg / mL, and containing no ascorbic acid. The culture was used under the condition of CO2.

< Experimental Example  1-2> Bone  Cytotoxicity Test on Cells

Cultured MC3T3-E1 was put into 1.5 x 10 ⁴ cells per well in a 96 well plate and incubated for 24 hours in 10% FBS-α-MEM medium. After removing the medium, and incubated in the α-MEM medium containing a composition containing the Harpagopitum root extract of Example 1 and the Harpagopitum root extract of Example 2 at a constant concentration, by performing MTT assay Cell viability was measured, and the results are shown in FIGS. 1A and 1B, respectively.

In Figure 1, the Harpagopitum root extract of Example 1 showed a cell viability of 80% or more when treated with the highest concentration of 500 μM concentration, the composition containing the Harpagopitum root extract of Example 2 Treatment with high concentration of 1000 ㎍ / mL showed cell viability of more than 70%

< Experimental Example  1-3> ALP  By dyeing Bone  Cell activity measurement

In this Experimental Example, the composition containing the Harpagopitum muscle extract of Example 1 and the Harpagopitum muscle extract of Example 2 was basic using MC3T3-E1 cultured to evaluate the effect on the activation of osteoblasts. The activity of phosphatase was measured. In general, basic phosphatase is known as a specific marker of early osteoblasts because its activity is clear at the early stage of osteoblast differentiation.

After cultured MC3T3-E1 to form a monolayer, it is treated with vitamin C and β glycerophosphate to induce differentiation into osteoblasts, Harpagopitum muscle extract of Example 1 and Harpagopitum muscle extract of Example 2 The composition containing the was cultured in α-MEM medium containing a constant concentration. As a comparative example, 17β-estradiol, currently used as a treatment for osteoporosis, was treated at a constant concentration. After 4 days, the cell layer was washed with phosphate saline, fixed in 3.7% formaldehyde and 90% ethanol for 2 minutes and washed in TBS for 10 minutes.

ALP staining was carried out using alkaline phosphatase (AP kit; Sigma Chemical Co., USA). First, the medium was removed and the fixed solution (citrate-acetone-formaldehyde) was used at room temperature for 30 seconds according to the manufacturer's instructions. It was stored and washed with distilled water for 45 seconds. The prepared diazonium solution (sodium nitrite: FRV-alkaline: naphthol AS-BI alkaline solution = 1: 1: 1) was added thereto, incubated at room temperature for about 15 minutes, washed with distilled water for 2 minutes, and hematoxylin solution (hematoxylin solution). After dyeing again for 2 minutes), the dye solution was washed thoroughly with running water, observed and photographed under a microscope (× 10), and the results are shown in FIGS. 2 and 3.

As shown in Figures 2 and 3, the untreated control group did not form much red enzyme, whereas the Harpagopitum muscle extract of Example 1 of the present invention was treated with the Harpagopi of Example 2 In the group treated with the composition containing the Tombeun extract, phosphatase activity was significantly increased in a concentration dependent manner.

< Experimental Example  1-4> AR  Examination of calcification formation by staining method

After inducing differentiation into osteoblasts in the same manner as in Experimental Example 1-3, the composition comprising the Harpagopitum muscle extract of Example 1 and the Harpagopitum muscle extract of Example 2 at a constant concentration Cultured in α-MEM medium. As a comparative example, 17β-estradiol, currently used as a treatment for osteoporosis, was treated at a constant concentration. The medium of the plate was completely removed, washed with PBS and fixed for 1 hour at 4 ° C. with 70% EtOH. Alizarine red solution was adjusted to 40 mM in 10 mL of distilled water, and calcium was dyed at pH 4.2, so the pH was adjusted. After fixation, the solution was dyed for 10 minutes and washed twice with distilled water. The undyed parts were washed with PBS and removed. The surface was soaked with PBS so that the surface was not too dry, and the degree of nodule formation was observed under a microscope.

The formation of nodules was dissolved in 10 mM sodium phosphate (pH 7.0) by adding 10% cetylpyridinium chloride for 15 minutes, and the degree of staining was measured by absorbance at 562 nm and is shown in FIGS. 4 and 5. .

As shown in Figures 4 and 5, osteoblast cells in a concentration-dependent manner in the group treated with the Harpagopitum root extract of Example 1 of the present invention and the composition containing the Harpagopitum root extract of Example 2 As the differentiation of the furnace proceeded, the absorbance values of the stained stains increased significantly.

< Experimental Example  2> Activation efficacy analysis of osteoclasts

< Experimental Example  2-1> osteoclast isolation

 The biggest difficulty in studying osteoclast differentiation is that no cell lines that maintain osteoclast function have been established to date. Since osteoclasts originate from hematopoietic stem cells of bone marrow origin and are assisted by osteoblasts / basal cells when they differentiate, the mutual culture system of bone marrow cells and osteoblasts is used as a differentiation model system of osteoclasts.

In the present invention, three weeks old male ICR mice were sacrificed and the tibias were isolated by disinfection with alcohol. Bone marrow was obtained by inserting a 26 gauge 1 mL syringe into the bone marrow cavity of the separated tibia and spraying α-MEM medium. Monocytes were isolated using Histopaque (Sigma).

< Experimental Example  2-2> Cytotoxicity Experiments on Osteoclasts

The isolated mouse bone marrow macrophages were put into 1.5 x 10 ⁴ cells per well in a 96 well plate and incubated for 24 hours in 10% FBS-α-MEM medium. After removing the medium, and incubated in the α-MEM medium containing a composition containing the Harpagopitum root extract of Example 1 and the Harpagopitum root extract of Example 2 at a constant concentration, by performing MTT assay Cell viability was measured and the results are shown in FIGS. 6 and 7, respectively.

6 and 7, when the composition containing the Harpagopitum root extract of Example 1 and the Harpagopitum root extract of Example 2 was treated at the highest concentration of 1000 μg / mL, the cell viability of 50% or more Indicated.

< Experimental Example  2-3> Analysis of the effect on the activity of osteoclasts

In order to see the effect on the osteoclast activity of the composition containing the Harpagopitum muscle extract of Example 1 and the Harpagopitum muscle extract of Example 2, the isolated and cultured mouse bone marrow macrophages were treated with 4 × 104 cells / The wells were aliquoted into 96 well plates. Induction of osteoclast differentiation after 24 hours of incubation was performed with 10% FBS a-MEM supplemented with 50 ng / ml of the osteoclast differentiation factor RANKL (RANKL). Cultured under conditions.

Treating the composition containing the Harpagopitum muscle extract of Example 1 and the Harpagopitum muscle extract of Example 2 prepared simultaneously with RANKL treatment, it is a marker of osteoclasts in the process of inducing progenitor cells into osteoclasts The effect of inhibiting the differentiation of osteoclasts was evaluated by measuring the enzyme activity of acid phosphatase.

Culture medium was removed from the culture plate with osteoclasts and treated with 10% formalin for 5 minutes to fix the cells. Formalin was removed and treated with 0.1% Trypton X-100 for 10 seconds.

Tryptone X-100 solution was removed and stained with TRACP (tartrate-resistant acid phosphatase) for 5 minutes. TRACP staining was used with the leukocyte acid phosphatase kit (LEUKOCYTE ACID PHOSPHATASE KIT, Sigma, cat.No. 387-A). The TRACP staining solution was removed, washed twice with distilled water and dried, and the number of TRACP positive multinucleated osteoclasts was counted with an optical microscope (X100), and the results are shown in FIGS. 8 and 9, respectively. 8, 9 when the number of 325 osteoclast cells of the control group was set to 100%, 78.2%, 68.9%, 40.9%, 16.6% of the Harpagopitum muscle extract of Example 1 and the above Example Treatment with the composition containing the Harpagopitum muscle extract of 2 decreased 82.2%, 71.4%, 53.2%, 8.3%.

< Experimental Example  3> Clinical histology of Harpagopitum muscle extract using animal model

The present inventors conducted an ovarian resection (Ovariectomy) to see how the composition containing the Harpagopitum muscle extract of Example 1 and the Harpagopitum muscle extract of Example 2 affect the treatment and prevention of osteoporosis Using osteoporosis-induced female rats (female rats) as an experimental model, histological observations were performed by cutting the vertebrae (L5) and the tibia in the ovarian ablation white rats.

< Experimental Example  3-1> Ovariectomy

100 mg / kg of ketamine (Ketamine) and 2% xyazine (Xylazine; Rompun 0.15 ml / kg) followed by general anesthesia followed by conventional hair removal and aseptic treatment (10% povidone-iodine scrub) followed by a 70 % alcohol wipe) was performed.

An incision about 1 ㎝ was made in the center of the abdomen, and care was taken not to damage major organs such as the diaphragm or liver. After ligating the ovary with a suture thread, ovarian ablation was performed on both sides. After ovarian resection, each organ was relocated into the abdominal cavity and layered sutures were performed with a suture thread. 50 ml / kg of antibiotic cefazolin was injected to prevent postoperative infection.

< Experimental Example  3-2> Administration of Harpagopitum Root Extract and Composition containing Harpagopitum Root

Drug doses for one rat were based on 250 g body weight. Specifically, the control white paper was administered only 30 ml of water per solid feed and horses, and the experimental group white paper containing a composition containing the Harpagopitum muscle extract of Example 1 and the Harpagopitum muscle extract of Example 2 per horse Oral administration at a constant concentration, every morning to prepare a daily amount of the drug and the remaining amount was not discarded, regardless of the remaining amount of the stock solution was added according to the number of water was always filled by the number of water. Be careful not to change the water bottle and check the number of animals every day and prepared a composition containing the Harpagopitum root extract of Example 1 and the Harpagopitum root extract of Example 2 according to the number of animals.

The extracts and compositions of the present invention were administered in the same manner as described above, and were bred while orally administering a composition containing the Harpagopitum muscle extract of Example 1 and the Harpagopitum muscle extract of Example 2 for 12 weeks. It was.

< Experimental Example  3-3> Weighing and Dietary efficiency

The weight was measured at intervals of 4 weeks at a certain time during the test period, the results are shown in FIG. As shown in FIG. 10, there was no significant difference in body weight between the control group and the experimental group. Therefore, there was no difference in growth due to the toxicity of the Harpagopitum root extract of Example 1 and the Harpagopitum root extract of Example 2 of the present invention.

< Experimental Example  3-4> Optical Microscopy of Tissue Specimens

Femoral bone tissue collected from rats to which the extract and the composition of the present invention were administered was demineralized in aqueous formic acid os fixed in 10% formarin solution. Observation site of bone tissue was cut with surgical knife and washed with xylene and paraffin embedding after dehydration process ranging from 20 to 100% alcohol and acetone. Paraffin embedded bone tissue was cut to 5 μm with micron, hematoxylin and eosin (H & E) staining and gomori staining were confirmed by micro CT.

11 and 12 show the results of measuring the three-dimensional fine sponges and cortical bone structure and indicators of the microstructure.

In the case of bone mass, the OVX group of the comparative example in FIG. 11 and FIG. 12 showed a 64.5% decrease in bone mass compared to the sham group, but the composition containing the Harpagopitum muscle extract of Example 1 and the Harpagopitum muscle extract of Example 2 In the group administered, it was confirmed that the bone mass increased by 309.9 and 549.3%, respectively. As a comparative example, the group treated with 17β-estradiol, which was used for treating osteoporosis, was used to excise the ovary and not receive the extract and the composition of the present invention (OVX group), the rat without the ovary (sham group) and the conventionally used osteoporosis treatment agent (E2). Group) appeared similar to the rat (sham group) that did not remove the ovary.

Also in the sponge bone thickness, as shown in Figures 11 and 12, the OVX group was 3.7% lower than the sham group, containing the Harpagopitum root extract of Example 1 and the Harpagopitum muscle extract of Example 2 In the group administered the composition was found to be 7.7%, 24.4% thicker than the OVX group.

The BMD of the femur was 38.1% lower in the OVX group than the sham group.

< Experimental Example  3-5> Blood bone biomarker   Content measurement

As a bone biomarker for measuring the treatment effect of osteoporosis according to the present invention, the concentration of calcium in the blood and the concentration of osteocalcin were determined. TRAP5b as an osteoclast activity index and ALP activity as an osteoblast activity index were measured.

At 12 weeks of the experiment, blood was collected after anesthesia with anesthesia. Blood collected from the heart was left at 4 ° C for 30 minutes, centrifuged at 3,000 rpm for 10 minutes to separate the serum, and the content of Ca in the serum was measured using a blood analyzer Ektachem DTⅡsystem. Osteocalcin was measured using OSTEOCALCIN MYRIA. Measurements were made using RIA kit (Techno genetics, 16099436), and the results are shown in FIGS. 13 and 14.

When comparing the Ca content in the serum, as shown in Figure 13, Figure 14, it showed statistically significant depending on the concentration of the extract and the composition of the present invention. Ca concentration of the OVX group did not show a significant difference from the sham group that did not remove the ovary, when the composition containing the Harpagopitum muscle extract of Example 1 and the Harpagopitum muscle extract of Example 2 No change in Ca concentration was observed after 12 weeks.

When comparing the content of osteocalcin in the serum, as shown in Figure 13, Figure 14, it showed statistical significance depending on the concentration of the extract and the composition of the present invention. In the OVX group, the concentration was significantly increased, and in the group to which the composition containing the Harpagopitum root extract of Example 1 and the Harpagopitum root extract of Example 2 was administered, the concentration was decreased.

The ALP activity, an osteoblast activity index, was significantly increased in the OVX group, and concentration-dependent in the group containing the composition containing Harpagopitum muscle extract of Example 1 and Harpagopitum muscle extract of Example 2 Decreased.

In the case of the osteoclast activity index TRAP5b activity, the OVX group was significantly increased compared to the sham group, and the group containing the composition containing the Harpagopitum muscle extract of Example 1 and the Harpagopitum muscle extract of Example 2 was administered. In concentration-dependently decreased.

Claims (7)

Harpagopitum root extract 100 parts by weight, bone chain beam 10 to 300 parts by weight, 50 to 500 parts by weight of glue, 50 to 500 parts by weight of Bokyoung, 10 to 300 parts by weight of raw sulfur and 10 to 300 parts by weight of ginseng extract Pharmaceutical composition for the prevention and treatment of bone metabolic diseases.
The method of claim 1,
The pharmaceutical composition for preventing and treating bone metabolic diseases has osteoblast proliferation, differentiation promoting activity, and osteoclast differentiation inhibiting activity, characterized in that the pharmaceutical composition for the prevention and treatment of bone metabolic diseases.
The method of claim 1,
The Harpagopitum muscle extract is a pharmaceutical composition for the prevention and treatment of bone metabolic diseases, characterized in that obtained by extracting the powder of the Harpagopitum muscle in water, lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof.
delete The method of claim 1,
The bone metabolic disease is a pharmaceutical composition for the prevention and treatment of bone metabolic diseases, characterized in that it includes osteoporosis, Paget's disease, periodontal disease, bone metastasis cancer or rheumatoid arthritis.
Bone metabolic disease comprising 100 parts by weight of Harpagopitum muscle extract, 10 to 300 parts by weight of bone chain beam, 50 to 500 parts by weight of glue, 50 to 500 parts by weight of Bokyeong, 10 to 300 parts by weight of fresh sulfur and 10 to 300 parts by weight of ginseng extract Dietary supplement for the prevention and improvement of human health.
The method according to claim 6,
A health functional food for preventing and improving bone metabolic diseases has osteoblast proliferation, differentiation promoting activity, and osteoclast differentiation inhibiting activity.

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