KR20110032446A - Composition comprising the extract of astragalus membranaceus bge. ,cinnamomum cassia and phellodendron amurensis for preventing and treating of osteoporesis and bone disease - Google Patents

Abstract

PURPOSE: A composition containing herb extract for preventing and treating osteoporosis and bone diseases is provided to suppress osteoclast activation and to promote the proliferation and differentiation of the osteoclast. CONSTITUTION: A pharmaceutical composition for preventing and treating osteoporosis contains mixed herb material extract of Astragalus membranaceus BGE, Cinnamomum cassia, and Phellodendron amurensis as an active ingredient. The extract is isolated using water, low alcohol of C1-C4, or mixture solvent thereof. The low alcohol is methanol or ethanol. A pharmaceutical composition and health food for preventing and treating bone diseases contains the mixed herb material extract as an active ingredient.

Description

Composition comprising the extract of Astragalus membranaceus BGE. ， Cinnamomum cassia and Phellodendron amurensis for preventing and treating of osteoporesis and bone disease}

The present invention relates to a composition for preventing and treating bone diseases, such as osteoporosis and periodontal disease, containing BHH10 ( Astragalus membranaceus BGE.), Cinnamomum cassia and Phellodendron amurensis extract.

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 the life-long process of bone remodeling, which is caused by the bones of osteoclasts in the bone marrow by hormones or physical stimuli applied to the bone. It is explained by the bone formation of bone matrix by osteoblasts that gathered at the site where bone resorption occurred by gathering and destroying bones on the surface (Park JS, Natl. Nutr., 215, pp 25-31, 2000).

Osteoblasts located on the bone surface have alkaline phosphatase (ALP), a glycoprotein enzyme in the cell membrane, and type I collagen (Col), osteocalcin (OCN), and osteopontin (OPN). , Secrete and calcify bone matrix substances such as bone sialoprotein (BSP) (Collier FM et al., Endocrinology, 139 (3), pp1258-1267, 1998). Bone maintains a balance of osteoblast and osteoclast activity, thereby maintaining a constant bone mass and regeneration of bone continuously occurs due to their activity. However, when osteoclast activity is increased than osteoblasts, bone mass decreases to induce osteoporosis, and postmenopausal women induce osteoporosis by decreasing blood estrogen concentration.

Osteoporosis is one of the most important social problems at present, which causes fractures of the pelvic bones due to osteoporosis, and in the United States, it causes about 260,000 women every year, of which about 12 to 20% The extent is death. In the context of aging society and active participation of women, fractures caused by osteoporosis and osteoporosis of the elderly and postmenopausal women cause serious problems.

Materials currently used for the treatment of osteoporosis include estrogen, androtic anabolic steroids, calcium preparations, phosphates, fluoride preparations, ipriflavones, and vitamin D ₃ . Estrogens inhibit osteoblastic cell death, increase cell survival, and promote osteoclast cell death to decrease cell survival, which is somewhat effective in treating menopausal symptoms and maintaining bone mineral density. There are side effects that cause back. Therefore, there are active researches around the world to develop selective estrogen receptor modulators (sERMs) that have only positive therapeutic effects of estrogens and to use them in the treatment of estrogen replacement. Attention is focused on the high estrogen receptor subtype ERβ. It is a drug that inhibits bone resorption and includes SERMs and fluoride. These drugs are effective in increasing the BMD for short-term treatment (~ 5 years), but do not promote the increase of bone mass with long-term administration. It causes side effects. Therefore, there is a need for the development of safe prophylactic and therapeutic agents that have a continuous effect of increasing bone mineral density even after long-term administration. In addition, the drug inhibits osteoporosis osteoclast activity and inhibits bone destruction or increases the activity of bone regeneration unit through the proliferation of osteoblasts. 1,25 (OH) ₂ D ₃ (calcitriol) (calcitonin), parathyroid hormone ( parathyroid hormone (PTH) and bisphosphonate preparations.

However, existing osteoporosis drugs have only the effect of blocking bone resorption or promoting bone formation and causing many side effects during long-term administration. Therefore, there is a need for the development of safe preventive and therapeutic agents that have a continuous effect of increasing bone mineral density even after long-term administration.

Astragalus membranaceus BGE. Is a perennial herb belonging to the family Fabaceae, growing in the mountains and commonly grown as a herb. 5 kinds of Korea, as well as in the Astragalus (Astragalus) Tamra Astragalus grows in Jeju (A. adsurgens), Astragalus often flowers, purple (A. dauricus), one that grows in the highlands of Mt. Paektu area Astragalus (A. uliginosus), etc. ( Iii) is growing. Astragalus, a herb used in Chinese medicine, is a long, cone-shaped, cone-shaped, or sticky, root of Astragalus. It is often branched to 10 to 80 cm in length and 0.2 to 15 mm in diameter. The outer surface is light white and there are several places of grayish brown cork layer. The surface is hard and the cut surface is fibrous, the vertical cut surface is white and the central part is sulfur yellow. Collected in spring or autumn and dried. As ingredients, colloids, mucus, starch, sucrose, glucose, fiber, vitamin B and the like are known. The nature is slightly warm, nonpoisonous and tastes sweet.

Astragalus is used as a herbal medicine in oriental medicine, mainly acts on the parenteral (폐), menopause (肺 經), as well as nerve (腎 經), large intestine (大腸 經) and trichophytes (三焦 經). The benefits of Astragalus are boil-boosting, high-marking (止汗 表 止汗: treatment to stop the sweat by strengthening the defense function of the skin), dihydrogen species (利 水 消腫) to swell the urine smoothly Therapeutic treatment to subside, and the takduk baeongwon (托 毒 排膿: using a drug that supplements the blood donation to assist the regular, to push out the venom to discharge the internal pus). Therefore, Astragalus is effective in chronic weakness, especially in weakness (脾胃 氣) weakness, there is also the excitatory effect of the central nervous system. Fatigue, helplessness, speech deterioration, pulsation (脈 軟 緩) If you have a symptom, such as good to take, long-term doses are effective. It is also used when there is a symptom of severe depression (中 氣 下陷: worsening of the function of the spleen) such as withdrawal, uterine disorder, visceral sewage, and uterine bleeding. It works by improving the constitution and increasing the tension of the whole body muscles. Peripheral nerve palsy, semi-incompetence that is a sequelae of stroke, chronic joint rheumatism, periarthritis of the joints, etc. These are pain or blood flow (는) caused by circulatory disorder due to Giheyangyang (氣血 兩 虛). From the point of view of blood travel, hemorrhagic hemorrhage (氣 行 則 血 行 治 血 先 治 氣) in terms of writing a supplement to the flag. Astragalus is a staple of medicine, so it is used for Pyojajahan (表 虛 自汗: lack of energy, the skin's defense function is not strong enough to sweat on its own). Used for nephritis, Astragalus is a diuretic that eliminates edema as well as lowers proteinuria and improves the nutrition of the whole body. Astragalus is used for long-time septic purpura. In modern medical terms, due to the antibacterial action and the body's increased resistance to self-drainage is easy to coalesce.

According to a recent study, Astragalus has been reported to be used as a prescription to help treat and recover cancer patients undergoing radiation therapy by boosting immunity and improving hematopoietic function (Cheng XD et al., Acta Biochim.Biophys. Sin. , 36 (3), pp211-217, 2004), and the results of promoting bone metabolism by oral Astragalus fraction extract in OVA rat model (Kim C et al) , Arch. Pharm. Res. , 26 (11), pp 917-924, 2003). However, there have been no teachings or disclosures of therapeutic effects due to inhibition of bone destruction, bone formation, and bone regeneration.

Cinnamomum cassia is a young branch of cassia (or cinnamon), an evergreen arboreal plant of the berry family, spicy, sweet, warm, and works on the heart, lungs, and bladder. It is known to strengthen the stomach, suppress paralysis, have analgesic and cardiovascular effects, expand the blood vessels of the skin and stimulate sweat glands to cause sweating, antipyretic effect, and inhibit the virus. Chills, fever, headache, body pain It is used for sweating or palpitations. Long columnar with many branches, 30-70 cm long, 0.3-1 cm thick. The surface is reddish brown or brown with vertical ridges and traces of fine wrinkles and small lumps of leaves and branches. The quality is solid, brittle and easy to cut, Guangxi and Guangdong provinces are mainly produced. Vietnam, Sri Lanka It is also grown in India. As a result of pharmacological experiments, it was found that sweating, antipyretic, analgesic, cardiopulmonary, antiallergic, antiviral and antifungal activity, but to date osteoporosis due to inhibition of bone destruction, bone formation and promoting bone regeneration No therapeutic effect has been taught or disclosed.

Hwangbaek ( Phellodendron amurensis ) is a term that refers to the bark of the yellow bark as a medicinal herb, also known as hwanggyeongpi . It is a deciduous broad-leaved arboreous tree, growing up to 10m in height. The leaves are opposite each other. It is a pinnate leaf, and the small leaflets are 5 to 13 sheets, and the egg-shaped lanceolate or long round shape. The leaf edge has small serrations and soft hairs, and the base of the middle vein has some long hairs. Yellow flowers bloom in May-June, which is a monolith of the male sex family. The cone-shaped stamen is a long circle, with five stamens in the male flower, and the degenerated stamens in the female flower are scaly. It has 5 rooms, with short pistils and 5 pistils. The fruit has a lot of berry and has a hard core in the center, and when rounded, it is purple-black. In Korea, it is distributed in all regions except Jeju and Jeonnam, and is distributed in Japan, Manchuria, China, Amur, and Usuri, and grows in scrub forests or valleys in the mountains.

Peel off the bark of the tree, remove the skin (조: coarse bark) or cut and dried in the sun, this is called baekbaek is used as a medicine and hypoglycemic action. In addition, pneumococcal pneumoniae, pneumococcal tuberculosis, staphylococcus, etc., while inhibiting the development of tumor cells, and inhibits the growth and sterilization. When taken, palpation of gastric juice is promoted by palpation of taste reflexes, which also leads to an increase in appetite. In addition, the general alkaloids do not have a systemic action, so even a large amount of administration does not have side effects, so can be used as a formal agent as well as a stomach agent. In addition, the drug does not produce resistance to various bacteria, so it can be used as a cleansing disinfectant when epidemic eye disease is epidemic. In addition, blood pressure lowering, central nervous system suppression, and anti-inflammatory effects have been reported.In oriental medicine, it is used for Hwangyeon Haedoktang, Shihocheonggantang, and Hyeonggaeyeongyotang, but to date osteoporosis by promoting bone formation and bone regeneration of yellow and white No therapeutic effect has been taught or disclosed.

In this regard, the present inventors have found that the mixed herbal medicine (BHH10) extract consisting of Astragalus, Gyeji, and Yellow and White extract increases the area of osseous bone, inhibits osteoclast activity, and increases osteoclast activity in osteogenic disease. The present invention has been completed by revealing that it can be effectively used in the treatment of bone diseases such as treatment and periodontal disease.

The present invention provides a composition for the prevention and treatment of osteoporosis, containing the mixed herbal medicine extracts of Astragalus, Caesar, and Yellow White, which have excellent bone fracture suppression, bone formation, and bone regeneration promoting effect, and prevention and treatment of bone diseases such as periodontal disease. will be.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of osteoporosis containing the mixed herbal medicine extract of Astragalus, Gyeji and yellow white as an active ingredient.

In another aspect, the present invention provides a pharmaceutical composition for preventing and treating bone diseases, containing the mixed herbal extracts of Astragalus, Gyeji and yellow white as an active ingredient.

In another aspect, the present invention provides a health food for preventing and improving osteoporosis, such as periodontal disease, containing the mixed herbal extracts of Astragalus, Gyeji and yellow white as an active ingredient.

In addition, the present invention provides a health food for preventing and improving bone diseases, such as periodontal disease, containing the mixed herbal extracts of Astragalus, Gyeji and Hwangbaek as an active ingredient.

The mixed herbal medicine extract of Astragalus, Cajun and Yellow and White of the present invention has the effect of restoring bone formation, inhibiting osteoclast activity, promoting osteoblast proliferation and differentiation, vascular endothelial growth factor (VEGF) secretion, bone in ovarian resection osteoporosis animal model By promoting the expression of BMP-2, which is a forming protein, it can be usefully used as a pharmaceutical composition for preventing and treating bone diseases, such as osteoporosis or periodontal disease, and health functional food.

Hereinafter, terms used in the present invention are defined.

As used herein, the term "osteoporosis" refers to a condition in which the amount of bone decreases and the bone strength decreases due to qualitative changes, so that fracture is likely to occur. The osteoporosis is meant to include both primary osteoporosis of primary osteoporosis and secondary osteoporosis of secondary osteoporosis.

As used herein, the term "bone disease" refers to any disease caused by decreased bone density. The bone disease is meant to include all of osteoporosis, bone formation insufficiency, periodontal disease or fracture.

As used herein, the term "periodontal disease" refers to all diseases in which plaque (gingiva bacterial membrane, plaque) and calculus formed by many bacteria always present in the mouth cause inflammation and destroy periodontal tissue. The periodontal disease is also commonly referred to as flavor, it means both gingivitis and periodontitis (periodontitis).

As used herein, the term "prevention" means any action that delays the development of bone disease or osteoporosis by administration of a composition of the present invention.

As used herein, the terms "treatment" and "improvement" refer to any action in which the symptoms of bone disease or osteoporosis improve or benefit from administration of the composition of the present invention.

As used herein, the term "administration" means providing a subject with any of the compositions of the present invention in any suitable manner.

The term "individual" as used in the present invention means any animal, such as human, monkey, dog, goat, pig or rat, which can improve the symptoms of bone disease or osteoporosis by administering the composition of the present invention.

As used herein, the term “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit or risk ratio applicable to medical treatment, which is the type, severity, and drug of the bone disease or osteoporosis in the individual. Activity, sensitivity to drug, time of administration, route of administration and rate of release, duration of treatment, factors including drug used concurrently, and other factors well known in the medical arts.

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for the prevention and treatment of osteoporosis or bone disease, containing a mixed herbal extract of Astragalus membranaceus BGE., Cinnamomum cassia and Phellodendron amurensis as an active ingredient.

The bone disease is preferably bone dysplasia or periodontal disease, but is not limited thereto.

The mixed herbal extract is preferably prepared by a method comprising the following steps, but is not limited thereto, and general extraction methods may be used:

1) extracting dried Astragalus membranaceus BGE., Cinnamomum cassia and Phellodendron amurensis as a solvent;

2) filtering the extracted extract; And

3) concentrating the filtered filtrate.

The Astragalus, Cinnamon and Yellow White can be used both grown and commercially available.

In the case of using them by mixing, it is preferable to use the mixture of Astragalus, Cinnamon and Yellow White in a weight ratio (w / w) of 1 to 5, respectively, but is not limited thereto. It is preferable that the compounding weight ratio of Astragalus: gage: yellow-white is 0.5-3: 0.5-3: 1, It is more preferable that the said compounding weight ratio is 2: 2: 1, It is not limited to this.

The solvent used for the extraction is preferably extracted using water, alcohol or a mixture thereof as a solvent, the lower alcohol is preferably C ₁ to C ₄ lower alcohol, the lower alcohol is ethanol or methanol Preferably, it is more preferred to use 30 to 70% ethanol, but is not limited thereto. The solvent is more preferably used but is not limited to a mixed solvent of water and ethanol having a mixing ratio (v / v) of about 1: 0.1 to 1:10, preferably 1: 0.2 to 1: 5.

The amount of the solvent used for the extraction is preferably to use about 1 to 20 times the weight of the mixed herbal medicine sample, and more preferably to extract about 2 to 10 times the sample weight, but is not limited thereto. The extraction temperature and time is preferably extracted for about 0.5 to 48 hours at room temperature, more preferably for 1 to 30 hours at room temperature, but is not limited thereto. The extraction is preferably performed once to five times, more preferably once to three times, but not always limited thereto. The extraction method is preferably carried out by any one extraction method selected from the group consisting of hot water extraction, cold needle extraction, room temperature extraction, reflux cooling extraction, ultrasonic extraction and steam extraction, but is not limited thereto. The extract can be used in any form, such as liquid, powder or concentrate.

 The mixed herbal medicine extract of the present invention is an indicator component as an indicator component is an organic extract of formononetin (formononetin), the ginseng extract is a cinnamic acid (cinnamic acid) and the yellowish white extract is a berberine (berberine), respectively, but is not limited thereto. Do not.

Specifically, the mixed herbal medicine extract of the present invention is 0.5 to 6 (w / w%) of formononetin, 0.2 to 2 (w / w%) of cinnamic acid (berberine), and berberine. It is preferable to include 0.5 to 5 (w / w%), formmononectin in the range of 1.0 to 3.00 (w / w%), cinnamic acid in the range of 1.0 to 0.3 (w / w%) w%), berberine is more preferably included in the range of 2.0 to 4.0 (w / w%), but is not limited thereto.

In one embodiment of the present invention, after washing the yellow, yellow and yellowish white, mixed with the same amount of each herbal medicine in a dried state, and then added 30% (v / v) ethanol aqueous solution of 8 to 10 times the sample The mixture was refluxed for 6 hours with stirring. The filtrate was collected and collected, and the residue was added to 30% (v / v) ethanol aqueous solution and re-extracted for 3 hours, and then the filtrates were combined and concentrated. Finally, the same amount of distilled water was added and suspended, followed by freeze drying to obtain a mixed herbal extract of powdered Astragalus, Caesar, and Yellow White (hereinafter referred to as 'BHH10 extract', a mixture of Astragalus, Caesar, and Yellow White). ).

In one embodiment of the present invention, in order to confirm that the BHH10 extract is a safe substance in vivo , body weight, tissue weight change by administration of BHH10 extract in ovarian isolated animal model, liver and Toxicity tests for kidneys, and blood count changes were measured. As a result, the BHH10 extract administration group of the present invention showed a similar increase in body weight and uterine weight to the qualitative group (Sham group), whereas Astragalus extract, cinnamon extract, yellow white extract, BHH17 (salt, cinnamon, purple, brown, yellow-white) , Perceptual mixed herbal medicine) extract and E2 (17β-estradiol) administration group was confirmed to increase the weight and weight of the uterus to the level of 1/2 (see FIGS. 4 and 5). In addition, there was no toxicity to the liver and kidneys, and there was almost no difference in blood cell count according to the administered drug (see Table 2).

In one embodiment of the present invention, in order to determine the effect of the BHH10 extract on the area of trabecula in vivo , the area of the tibial bone of the tibia using a quantitative image analysis system As a result of the calculation, the negative control group extracted from the ovary was found that the osteoporosis was induced by reducing the area of the soju bone by 45% compared to the normal group (Sham group), and after the administration of the BHH10 extract of the present invention, It was found to increase by 191% compared to the control. This can be seen that the astragalus extract, gyeji extract, baekbaek extract, BHH17 and E2 (17β-estradiol) administration group significantly increased the bone area (see Fig. 6 and Table 3).

In one embodiment of the present invention, in order to determine the effect of BHH10 extract on bone density in vivo, the bone density change of the tibia, lumbar spine, and femur was measured using a bone density meter, and the ovarian extracted negative control group was the Sham group (normal Bone densities of tibia, lumbar spine, and femur were decreased in the tibia, lumbar spine, and femur, and there was no significant change in Astragalus extract, Cage extract, Yellow white extract and BHH17 extract. It was confirmed to increase (see FIG. 7).

In one embodiment of the present invention, in order to determine the effect of the BHH10 extract on blood hormone in vivo, as a result of quantifying the blood hormone, the negative control group extracted ovary reduced GH (growth hormone), Astragalus extract, Caji extract , Bleach extract, BHH17 extract and BHH10 extract were not significantly affected by the administration of the drug, E2 administration group showed a significant increase in growth hormone (see Fig. 8).

In one embodiment of the present invention, in order to determine the effect of the BHH10 extract on the change in the expression of bone formation-related factors in vivo, the measurement of the bone growth-related growth factor (VEGF) and bone-forming protein (BMP-2) in the blood As a result, the ovarian-extracted negative control group had decreased VEGF and BMP-2 proteins, but the BHH10 extract showed an increase in bone formation protein factors that were superior to the Astragalus extract, Cajun extract, Bleach extract, BHH17 extract and E2 administration group (see Fig. 9). In addition, the results of measuring osteocalcin (OCN), BHH10 extract showed a more excellent increase than the Astragalus extract, Cinnamon extract, yellow white extract and BHH17 extract, and showed the same level of expression as the E2 administration group (see Fig. 10).

Therefore, the mixed herbal medicine extracts of Astragalus, Cajun and Astragalus of the present invention are extracts of Astragalus Astragalus, Caesaric extract, Astragalus extract, Astragalus, Astragalus, Licorice, Brown root, Astragalus, Persimmon extract, and E2 (17β-) Compared to estradiol), the area of the subcetabular bone is significantly increased, and it can be seen that it can be usefully used as a therapeutic agent for osteoporosis or bone disease by confirming that it is a safe substance compared to the above substances.

In one embodiment of the present invention, in order to determine whether BHH10 extract has an activity inhibitory effect on osteoclasts of subchondral bone of human, a tartrate-resistant acid phosphatase (TRAP) staining method is used. As a result of the measurement, it was confirmed that the BHH10 extract of the present invention inhibited the number of reacted osteoclasts in a concentration-dependent manner (see FIG. 11).

In one embodiment of the present invention, to determine whether BHH10 extract has an activity promoting effect on the proliferation of osteoblasts in the subchondral bone of human, measured by using the WST-8 assay method, BHH10 of the present invention It was confirmed that the extract significantly increased osteoblast survival of subchondral bone tissue (see FIG. 12).

In one embodiment of the present invention, in order to determine whether BHH10 extract affects osteoblast differentiation, the activity of alkaline phosphatase (ALP) was measured in osteoblasts of subchondral human tissue. BHH10 extract showed an excellent effect of increasing the ALP activity was found to promote osteoblast differentiation of human subchondral bone tissue (see Fig. 13).

In one embodiment of the present invention, to determine whether the BHH10 extract proliferates osteoblast growth, the effect on the proliferation of Saos-2 cells, osteoblast-like cells, is used in the WST-8 assay method. As a result of the measurement, the BHH10 extract of the present invention did not affect the survival rate of Saos-2 cells in the range of 100 ㎍ / ㎖ concentration or less, it was confirmed that it is a safe drug without cytotoxicity, significantly saos-2 cell survival It was confirmed to increase (see FIG. 14).

In one embodiment of the present invention, in order to determine whether BHH10 extract affects osteoblast differentiation, the activity of alkaline phosphatase was measured in Saos-2 cells, which are osteoblast-like cells. It can be seen that the BHH10 extract promotes osteoblast differentiation by showing an excellent effect of increasing ALP activity (see FIGS. 15 and 16).

In one embodiment of the present invention, in order to determine the effect of BHH10 extract on the expression of vascular endothelial growth factor (VEGF), the expression level of VEGF in osteoblasts using ELISA kit and RT-PCR As a result, it was confirmed that the BHH10 extract of the present invention increased the secretion of VEGF, an angiogenesis-promoting factor (see FIGS. 17 and 19).

In one embodiment of the present invention, in order to determine the effect of BHH10 extract on the expression of bone morphogenic protein-2 (BMP-2), the expression level of BMP-2 in osteoblasts was measured using an ELISA kit. As a result, it was found that the BHH10 extract of the present invention promotes bone formation by significantly increasing BMP-2 secretion even at low concentrations (see FIGS. 18 and 20).

In one embodiment of the present invention, whether BHH10 extract affects the activity of osteocalcin (OCN), osteopontin (OPN) and type I collagen (type I collagen, Col I), which are related to bone formation In order to analyze the expression level of OCN, OPN and Col I genes by RT-PCR, RNA was extracted from osteoblast-like cell lines. As a result, it was found that the BHH10 extract of the present invention promotes the activity of bone-related genes. (See FIGS. 21 and 22).

In one embodiment of the present invention, in order to determine the effect of BHH10 extract on osteoblast differentiation and bone formation, intracellular calcium deposition in osteoblast-like cell lines was analyzed by staining method. The concentration of calcium in the cells can be confirmed by quantitatively measuring the degree of calcium deposition (see FIGS. 23 and 24).

Therefore, the BHH10 extract of the present invention has a osteogenic restorative effect, inhibits the activity of osteoclasts, promotes the proliferation and differentiation of osteoblasts, increases the growth factor and osteogenic protein expression, and has little toxicity and side effects. It can be seen that it can be usefully used as an active ingredient for preventing and treating osteoporosis or bone disease.

BHH10 extract of the present invention may contain 0.1 to 50% by weight of the BHH10 extract for the prevention and treatment of osteoporosis or bone disease.

Pharmaceutical compositions comprising the BHH10 extract of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

The pharmaceutical compositions comprising the BHH10 extract of the present invention may be in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, suppositories, and sterile injectable solutions, respectively, according to a conventional method. Can be formulated and used. Carriers, excipients and diluents which may be included in the composition comprising the extract of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate , Calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations include at least one excipient such as starch, calcium carbonate, sucrose in the extract. Or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The preferred dosage of the BHH10 extract of the present invention depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the extract of the present invention may be administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 100 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

The BHH10 extract of the present invention can be administered to mammals such as mice, mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example, by oral, rectal, intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

The present invention also provides a method for treating bone disease, comprising administering to a subject suffering from osteoporosis a composition comprising a pharmaceutically effective amount of a BHH10 (mixed medicinal herb of Astragalus, Caesar, and Yellowish White) extract of the present invention.

The present invention also provides a method for preventing osteoporosis, comprising administering to a subject a composition comprising a pharmaceutically effective amount of a BHH10 extract of the present invention.

The present invention also provides a method for treating bone disease, comprising administering to a subject suffering from a bone disease a composition comprising a pharmaceutically effective amount of a BHH10 (mixed medicinal herb of Astragalus, Caesar, and Yellow White) extracts of the present invention. .

The present invention also provides a method for preventing bone disease, comprising administering to a subject a composition comprising a pharmaceutically effective amount of a BHH10 extract of the present invention.

The bone disease is preferably bone dysplasia or periodontal disease, but is not limited thereto.

BHH10 extract of the present invention shows an excellent osteogenic restorative effect in ovarian resection osteoporosis animal experiments, by inhibiting the activity of osteoclasts, promote the proliferation and differentiation of osteoblasts, increase the growth factor and osteogenic protein expression, osteoporosis Or it can be usefully used for the prevention or treatment of bone diseases, such as periodontal disease.

In addition, the BHH10 extract of the present invention is significantly compared to the extract of Astragalus extract, Cinnamon extract, yellowish white extract, and Astragalus, Cinnamon, Licorice, brown root, yellow white, crust mixed herbal medicine, and E2 (17β-estradiol), which is a conventional osteoporosis therapeutic agent. It showed a high osteogenic effect, it can be seen that it can be usefully used as an active ingredient in the treatment of osteoporosis or bone disease by confirming that it is a safe substance compared to the above substances.

In the administration of the present invention, the composition containing the BHH10 extract may further comprise a suitable carrier, excipient or diluent according to conventional methods.

In the administration of the present invention, the composition of the present invention is formulated in the form of an oral dosage form such as powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol, external preparation, suppository or sterile injectable solution according to a conventional method. Can be used. More specifically, when formulated, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories.

In the administration of the present invention, the composition of the present invention is possible in any manner of administration, and may be administered by oral, rectal, intravenous, intraperitoneal, intramuscular or subcutaneous injection, for example.

In the administration of the present invention, the preferred dosage of the composition of the present invention depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. It may be administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 100 mg / kg per day. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

In addition, the present invention provides a health functional food for the prevention and improvement of osteoporosis or bone disease containing BHH10 (mixed herbal medicine of Astragalus, Gyeji and yellow white) as an active ingredient.

"Health functional food" as defined in the present invention means a food manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to Act No. 6767 of the Health Functional Food Act, and "functional" means a human body It means the ingestion for the purpose of obtaining a useful effect in health use such as nutrient control or physiological action on the structure and function of.

Foods to which the BHH10 extract can be added include, for example, various foods, beverages, gums, teas, vitamin complexes, health functional foods, powders, granules, tablets, capsules or beverages.

It may also be added to food or beverages for the purpose of preventing and improving osteoporosis or bone disease. At this time, the amount of the extract in the food or beverage may be added in 0.01 to 15% by weight of the total food weight, the health beverage composition may be added in a ratio of 0.02 to 5 g, preferably 0.3 to 1 g based on 100 ml. have.

The health functional beverage composition of the present invention is not particularly limited to other ingredients except for containing the BHH10 extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. . Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (tauumatin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the BHH10 extract of the present invention is used in various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, such as flavoring agents, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and Salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the BHH10 extract of the present invention may contain flesh for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the BHH10 extract of the present invention.

Hereinafter, the present invention will be described in detail by the following Examples, Comparative Examples, Experimental Examples and Preparation Examples.

However, the following Examples, Comparative Examples, Experimental Examples, and Production Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples, Comparative Examples, Experimental Examples, and Production Examples.

Example 1 Preparation of Mixed Herbal Medicine (BHH10) Extract

Kwanghee Medical Center Purchased Astragalus, Gyeji, and Hwangbaek from Oriental Medicine Hospital, washed with water to remove contaminants, and dried each herb in a well-dried state in which the weight ratio of Astragalus, Gyeji, and Hwangbaek was 2: 2: 1. 1 g of 30% (v / v) ethanol aqueous solution was added to the g, and the mixture was refluxed for 6 hours with stirring. The filtrate was collected and collected. The residue was re-extracted by adding 0.5 L of 30% (v / v) aqueous ethanol solution for 3 hours, and the filtrates were combined and concentrated to 0.2 L. Finally, the same amount of distilled water was added and suspended, followed by freeze-drying, to obtain a mixed herbal extract (yield: 17.8%) of powdered Astragalus, Caesar, and Yellowish White. The 'medicinal herb extract of Astragalus, Gyeji, and Hwangbaek' was named 'BHH10 extract'.

Comparative Example 1 Preparation of Astragalus Extract

100 g of Astragalus, purchased from Kyunghee Medical Center, was rinsed and dried. Then, 1 L of a 30% (v / v) ethanol aqueous solution was added thereto, followed by boiling extraction at 85 ° C. for 6 hours while stirring. After filtration, concentrated to dryness, 27 g of Astragalus extract was obtained and used as a sample of the following experimental example.

As a result of chromatogram analysis using the extract extracted and purified by the above method, as shown in FIG. 1, the formononetin content was 1.3% (FIG. 1).

Comparative Example 2 Preparation of Cinnamon Extract

100 g of gyeji purchased from Kyung Hee Medical Center was rinsed and then rinsed. Then, 1 L of 30% (v / v) ethanol aqueous solution was added thereto, followed by boiling extraction at 85 ° C. for 6 hours while stirring. After filtration, concentrated to dryness, 8.1 g of the extract was obtained and used as a sample of the following experimental example.

As a result of chromatogram analysis using the extract extracted and purified by the above method, as shown in FIG. 2, the trans-cinnamic acid content was 1.3% (FIG. 2).

Comparative Example 3 Preparation of Yellow-White Extract

100 g of yellow white purchased from Kyung Hee Medical Center was dried in the shade and then rinsed. Then, 1 L of 30% (v / v) ethanol aqueous solution was added thereto, followed by extraction with boiling water at 85 ° C. for 6 hours while stirring. After filtration, concentrated to dryness, 17.9 g of a yellowish white extract was used as a sample of the following experimental example.

As a result of chromatogram analysis using the extract extracted and purified by the above method, as shown in FIG. 3, the berberine content was 2.6% (FIG. 3).

Comparative Example 4 Preparation of BHH17 Extract

After washing the yellow, yellow, red, brown, yellow, and crust, which were purchased at Kyunghee Medical Center, remove the contaminants and make them dry, and then make each herbal medicine yellow, yellow, red, yellow, white, crust. The mixture was mixed at a ratio of 2: 2: 2: 2: 1: 2, and 1L of a 30% (v / v) ethanol aqueous solution was added to 100 g, followed by extraction under reflux for 6 hours with stirring. The filtrate was collected, and the residue was re-extracted for 3 hours by adding 0.5 L of 30% (v / v) ethanol aqueous solution, and the filtrates were combined and concentrated to 0.2 L. Finally, the same amount of distilled water was added, suspended and freeze-dried to obtain a powdered BHH17 extract (amount: 25.9%).

Example 2 Preparation of Ovarian Extracted Rat (OVX) Animal Model

Sprague Dawley rats of 200-300 g body weight obtained from Kyunghee Medical Center were subjected to bilateral ovarian extraction in all rat females of the control group and the test group except the sham group (normal group). Both ovaries were resected (OVX group) or all operations except ovarian resection (Sham group) followed by a one week recovery period followed by nine weeks of administration. The test samples were administered orally or intraperitoneally to the OVX group by dose and period, and 17beta-estradiol, a therapeutic agent for osteoporosis, is currently used as a comparative material for the samples. Total blood counts were collected by preoperative administration, before administration, and every administration period. Plasma was isolated and used for subsequent experiments. After a period of administration, organ tissues, tibia, femur, and lumbar spine were separated and subjected to toxicity, morphometric, and BMD measurements.

Example 3 Culture of Human Osteoclastus Cells in Subchondral Bone

At Kyung Hee Medical Center, the patients with osteoarthritis underwent surgery of the subchondral bone of the subchondral tissue obtained from normal femoral tissue during surgery. The bones of the subchondral bone of the lower tissue were crushed into pieces and treated with type II collagenase for 30 minutes to separate them. Cells were transformed into α-MEM (alpha-minimal essential medium) with 10% FBS, 25 ng / ml M-CSF, 25 ng / ml + 25 ml / M-CSF, 25 ng / ml sRANKL 25 ng / ml + M-CSF The cells were differentiated into osteoclasts in each medium composition containing 25 ng / ml + VEGF 50 ng / ml.

Example 4 Culture of Osteoblasts of Human Subchondral Cartilage

At Kyung Hee Medical Center, the patients with arthritis patients underwent surgical treatment of the subchondral bone of the subchondral tissue obtained from the normal femur. The bones of the subchondral lower tissue were crushed into pieces and treated with type II collagenase for 30 minutes, followed by explant culture. After culturing in DMEM medium to which 10% FBS was added, it was used after subcultured twice.

Example 5 Osteoblast Cell Culture

Saos-2 cells (ATCC accession no .; HTB-85), a human-derived osteoblast-like cell line, were obtained from the American Type Culture Collection (ATCC), 15% fetal bovine serum (FBS) and 1 Culture was performed using medium (McCoy's 5A, Gibco BRL, USA) to which% penicillin / streptomycin (Invitrogen Corporation, CA, USA) was added.

Example 6 Reverse Transcription Polymerase Chain Reaction (RT-PCR)

RNA was prepared using Trizol reagent (TRIzol reagent, Invitrogen Corporation, CA, USA). Reverse transcription by adding buffer to 1 μg total RNA, Oligo (dT) ₁₂ primer, dNTP (10 mM), 0.1M dithiothritol (DTT, dithiotreitol), reverse transcriptase (Promega, USA) and RNase inhibitor The reaction was carried out at 42 ° C. for 60 minutes. Polymerase chain reaction (PCR) using each of the cDNAs synthesized above and the primers described in [Table 1] is 0.5 μg of cDNA and 2.5 units of Taq polymerase (TaKaRa Taq ^™ , Takara, Japan), Mix 1.5 mM dNTP, 1 × buffer (10 mM Tris-HCl pH 8.3, 50 mM KCl, 0.1% Triton X-100), 20 pmol of each primer pair, adjust the total volume to 10 μl with distilled water, and then use a machine (TaKaRa Biotechnology, Seoul, Korea). The product produced by PCR was subjected to electrophoresis on 1.8% agarose gel and stained with EtBr (ethidium bromide) to identify a specific band. PCR product beta-actin (β-Actin) was used as an indicator gene, and quantified using Gel-Doc EQ (BIO-RAD Laboratories, Milan, Italy).

gene Sequence of primer β-actin Forward (SEQ ID NO: 1) 5'-CCTGGCCAAGGTCATCCATGACAACTTTGG-3 ' Reverse (SEQ ID NO: 2) 5'-CATGAGGTCCACCACCCTGTTGCTGTAGCC-3 ' OCN Forward (SEQ ID NO: 3) 5'-GAGGACCCTCTCTCTGCTCACTCTGCTGGC-3 ' Reverse (SEQ ID NO: 4) 5'-GTGGTGCCATAGATGCGCTTGTAGGCGTCC-3 ' OPN Forward (SEQ ID NO: 5) 5'-CTCGGATGAATCTGACGAATCTCACCATTC-3 ' Reverse (SEQ ID NO: 6) 5'-CTTACTCTTAGGGTCTAGGACTAGCTTGTC-3 ' Coll Forward (SEQ ID NO: 7) 5'-TGGTCTTGGAGGAAACTTTGC-3 ' Reverse (SEQ ID NO: 8) 5'-AGTATCTCCTTTGGCACCATC-3 ' VEGF Forward (SEQ ID NO: 9) 5'-TGCACCCACGACAGAAGGGGA-3 ' Reverse (SEQ ID NO: 10) 5'-TCACCGCCTTGGCTTGTCACAT-3 ' BMP-2 Forward (SEQ ID NO: 11) 5'-CAGCCATGAATTTCACAGCC-3 ' Reverse (SEQ ID NO: 12) 5'-GGGAGTTTCCATGAAGCCAC-3 '

Experimental Example 1 Measurement of Changes in Body Weight and Tissue Weight by BHH10 Extract Administration in Ovarian Animal Models

<1-1> Changes in body weight and tissue weight

After administering the BHH10 extract, Astragalus extract, Cinnamon extract, Yellow white extract and BHH17 extract to the experimental animal from which the ovary was extracted according to <Example 2>, the weight of the administered experimental animal was measured every week, and for a predetermined period of time. After administration, the tissue weight was measured, and the body weight and tissue weight of the Sham group and the control group were compared by a boneferroni multiple comparison method.

As a result, as shown in Figure 4, the control group showed a sharp increase in body weight compared to the Sham group, the group administered with 5 g / kg / day of BHH10 extract showed a similar weight gain as the control group. The control drug group (E2) showed a moderate weight gain phenomenon between the Sham group and the control group (FIG. 4).

In addition, as shown in Figure 5, the weight of the uterus was sharply reduced in the negative control group compared to the Sham group (normal group), Astragalus extract, bastard extract, yellow white extract, BHH17 extract and E2 administration group is 1/2 of the normal group Recovery above level. The BHH10 extract 5g / kg / day administration group was not different from the control group (Fig. 5).

Therefore, BHH10 extract does not change the weight and weight of the uterus compared to the control and control drug group, it is judged to be safe without the hormone changes in the body.

<1-2> Total Blood Cell Count and Organ Toxicity Measurement

After administering the BHH10 extract, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4 extract and the positive control group (Estradiol; E2), respectively, to the experimental animal from which the ovary was extracted according to Example 2, Toxicity test for liver and kidney by long-term administration of the drug showed that the dose in this study did not show toxicity to liver and kidney. Complete blood cell counts (CBC) measurements also showed that WBC, LY, MO, and GR decreased at the end of all groups compared to preoperative (10 weeks old) and post-test (20 weeks old). However, there was no difference according to the drug administered at each age (Table 2).

Age
(week) Control Sham Comparative Example 1 BHH10 E2 (positive control) WBC 10 18.8 ± 1.3 16.6 ± 2.5 15.3 ± 1.6 16.6 ± 1.3 20.6 ± 1.9 20 7.9 ± 0.8 6.0 ± 0.5 6.5 ± 0.7 7.7 ± 0.8 9.1 ± 1.1 LY 10 14.3 ± 1.0 12.2 ± 2.0 12.1 ± 1.1 11.8 ± 1.1 16.9 ± 1.6 20 6.5 ± 0.6 4.9 ± 0.4 5.3 ± 0.5 6.4 ± 0.7 7.1 ± 0.9 MO 10 2.6 ± 0.3 1.9 ± 0.3 1.8 ± 0.4 2.3 ± 0.3 2.2 ± 0.2 20 0.9 ± 0.1 0.7 ± 0.1 0.9 ± 0.2 1.0 ± 0.2 1.0 ± 0.1 GR 10 2.7 ± 0.3 2.5 ± 0.4 1.4 ± 0.3 2.6 ± 0.3 3.2 ± 0.6 20 0.6 ± 0.2 0.4 ± 0.1 0.4 ± 0.1 0.4 ± 0.1 0.9 ± 0.3 RBC 10 7.47 ± 0.10 7.93 ± 0.19 7.60 ± 0.17 7.58 ± 0.16 7.68 ± 0.12 20 8.29 ± 0.08 7.94 ± 0.19 7.58 ± 0.20 8.09 ± 0.08 8.39 ± 0.12 Hb 10 13.8 ± 0.3 14.3 ± 0.2 14.1 ± 0.1 14.0 ± 0.2 14.1 ± 0.2 20 15.1 ± 0.1 14.2 ± 0.3 14.0 ± 0.2 14.9 ± 0.2 15.4 ± 0.2 HCT 10 42.1 ± 0.5 44.2 ± 0.8 43.3 ± 0.8 43.2 ± 0.8 43.6 ± 0.5 20 46.3 ± 0.4 43.5 ± 0.8 42.7 ± 1.0 45.3 ± 0.3 46.7 ± 0.6

Experimental Example 2 Measurement of the Bone Histology by BHH10 Extract in Ovarian Animal Models

After the administration of the BHH10 extract, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4 extract and the positive control group (Estradiol; E2) to the experimental animal extracted ovary by <Example 2> After dissection, the lumbar and tibial tissues were separated.

After lumbar and tibial tissues were fixed in 10% formalin solution, demineralized in formic acid, the tissues were cut off, and then dehydrated to xyled by a stepwise dehydration process ranging from 70% to 100% alcohol and acetone. Clear and paraffin artery was performed. Paraffin-embedded tissue was cut into 5 microns with a microtome, followed by hematoxylin and eosin (H & E) staining, followed by optical microscopy, followed by morphometric analysis.

The quantizer of the Quantitative image analysis system (Wild Leits Co.) draws along the contours of each shochu and calculates the area, which automatically calculates the area of the subcetabular bone, which allows the growth plate to proximal to each tibia. The area of trabecula in the inside of the rectangle having a reference area of 2 × 10 6 μm ² was measured at a length of about 2/3 of the length of the growth plate in the lower part of the.

As a result, as shown in FIG. 6, the negative control group extracted from the ovary was found to have an osteoporosis induced by 45% of the area of the subcetabular bone compared to the Sham group (normal group), Astragalus extract, Gyeji extract, Yellow white extract, After administration of the BHH10 extract of the present invention compared to the BHH17 extract and the E2 administration group, the small bone area was increased (P <0.05) (FIG. 6).

In addition, in the following [Table 3], as shown in the Nanan bar 소 BHH10 extract treatment group increased the area of the tibial bone of the tibia up to 191% of the control group was significantly superior to the E2 administration group (Table 3).

Tibial Small Injury Area Lumbar bone minor bone area Control group 100.0 ± 13.2 100.0 ± 5.4 Sham County 243.5 ± 10.7 ^＃ 116.4 ± 5.9 Astragalus Extract 169.1 ± 16.2 110.1 ± 2.3 Cage Extract 164.2 ± 14.7 109.7 ± 4.6 Reed extract 157.7 ± 18.9 106.4 ± 5.9 BHH17 Extract 164.2 ± 14.7 ^* 111.2 ± 8.9 BHH10 Extract 218.6 ± 10.0 ^* 104.5 ± 2.1 E2 (positive control group) 160.9 ± 13.2 ^* 108.1 ± 1.1

＃: p <0.05 compared with negative control; And *: p <0.05 compared with Sham group.

Experimental Example 3 Measurement of Bone Mineral Density and BHH10 Extract in Ovarian Animal Models

In the animal experiment of Example 2, tibia, lumbar spine, and femur were separated, and bone density was measured using a bone density meter (LUNAR PIXImus), and based on these, changes in bone density for each drug were calculated.

As a result of the experiment, the negative control group extracted from the ovary had a bone mineral density (BMD) of the tibia, lumbar spine, and femur as compared with the Sham group (normal group), which was significantly reduced, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4 There was no significant change in the extract, BHH10 extract and E2 administration group was confirmed that the bone density in the tibia and lumbar bone significantly increased compared to the control group (Fig. 7).

Experimental Example 4 Measurement of Changes in Bone Formation Related Factors by BHH10 Extract Administration in Ovarian Animal Models

After administering the BHH10 extract, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4 extract and the positive control group (Estradiol; E2), respectively, to the experimental animal from which the ovary was extracted according to Example 2, As a result of hormone quantification, GH (growth hormone) was decreased by ovarian removal in the control group, but Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4 extract and BHH10 extract were significantly increased by administration of the drug. Although not affected, the E2 administration group showed a significant increase in growth hormone (FIG. 8).

In addition, as a result of measuring blood growth-related bone growth-related growth factors (VEGF) and bone formation proteins (BMP-2), VEGF and BMP-2 proteins were decreased by ovarian removal in the control group. Compared with Comparative Example 2, Comparative Example 3, Comparative Example 4 and E2 administration group, bone formation and protein factors were superior to those of the E2 administration group (Fig. 9).

In addition, BHH10 extract showed an increase in comparison with Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4 extract, and showed the same level of expression as the E2 administration group (Fig. 10). .

In conclusion, BHH10 extract of the present invention did not increase body weight and toxicity in organs in osteoporosis animal model, was not affected by growth hormone, and increased bone mineral density and bone density due to increased expression of growth factor and bone formation protein factor. It has been shown that the effect of increasing the.

Experimental Example 5 Determination of the Inhibitory Effect of the Mixed Herbal Medicine (BHH10)

The inhibitory effect of the BHH10 extract of the present invention on the osteoclasts of the subchondral bone of human was measured by using a tartrate-resistant acid phosphatase (TRAP) staining method (Leukocyte Acid Phosphatase kit, Sigma, MO, USA).

Specifically, the osteoclasts of <Example 3> in each well (well) to be 1 × 10 ⁵ cell number / well in a 48-well plate (well plate), and then cultured, BHH10 extract by concentration ( 100 μl each of Control, 0.1, 1, 10 μg / ml) was incubated for 7 days. After removing all the culture medium and fixing the cells in a fixed solution (fixative solution) for 30 seconds, using a TRAP staining kit using acid phosphatase activity that is characteristic of osteoclasts Osteoclasts were stained.

As a result, it was confirmed that the BHH10 extract inhibited the number of osteoclasts positively reacted in TRAP staining as shown in FIG. 11 (FIG. 11).

Experimental Example 6 Measurement of Osteoblast Growth of Subchondral Bone of BHH10 Extract

The effect of BHH10 extract of the present invention on the proliferation of osteoblasts in human subchondral bone tissue was measured using the WST-8 assay (cell counting kit-8, Dojindo molecular technologies, Tokyo, Japan).

Specifically, after dispensing osteoblasts of <Example 4> in 96-well plate at a concentration of 1 × 10 ⁴ cells / well, BHH10 extract by concentration (0.01, 0,1, 1, 10, 100 Μg / ml) and then 100 μl were incubated for 72 hours. 10 μl of WST-8 (water-soluble tetrazolium salt; 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium, monosodium salt) After addition and incubation for 2 hours, when WST-8 formazan was formed and developed, the absorbance was measured and measured at 450 nm using an ELISA reader. Cell viability was expressed as a percentage of the absorbance of the sample treated group relative to the control group.

As a result, as shown in FIG. 12, it was confirmed that BHH10 extract significantly increased osteoblast survival of subchondral bone tissue for 3 days in the range of 0-100 μg / ml concentration (FIG. 12).

Experimental Example 7 Measurement of Alkali Phosphatase (ALP) Activity in Osteoblasts of Human Subchondral Bone of BHH10 Extract

Alkaline phosphatase (ALP), a serum enzyme, is an enzyme that is an indicator of bone formation and is produced during the differentiation of osteoblasts (Zhao Y et al., Biol. Pharm. Bull. , 28 (8), pp1371-1376). , 2005).

In this regard, ALP activity was measured to determine whether BHH10 extract affects osteoblast differentiation. ALP activity is the degree of the development of intracellular ALP by decomposing para-nitrophenylphosphate (p-nitrophenylphosphate (pNPP) into para-nitrophenol and phosphate) and developing the kit (ALP kit, Sigma-Aldrich Co. , MO, USA).

Osteoblasts prepared by treatment as described in <Experimental Example 4> were lysed with 0.1% Triton X-100 (Triton X-100, Sigma, USA), and then treated with an ultrasonic machine (Branson, USA) at 14,000 g and 4 ° C. Centrifuged for 20 minutes. The supernatant was incubated for 3 minutes at 37 ° C with a reaction buffer, and then the reaction was stopped using 0.1 N NaOH, and the absorbance was measured at 405 nm. The standard line was quantified using para-nitrophenol (p-nitrophenol; Sigma-Aldrich Co., MO, USA), and based on this, the ALP amount of the sample was converted.

Experimental results, as shown in Figure 13, BHH10 extract of the present invention, after 3 days of treatment to osteoblasts, it was confirmed that the excellent ALP activity at 0.01, 0.1, 1 and 10 ㎍ / ㎖, BHH10 extract of human It was found to promote osteoblast differentiation of subchondral bone tissue (FIG. 13).

Experimental Example 8 Measurement of Osteoblast Growth Proliferation Effect of BHH10 Extract

The effect of BHH10 extract on the proliferation of Saos-2 cells, osteoblast-like cells, was measured using the WST-8 assay (cell counting kit-8, Dojindo molecular technologies, Tokyo, Japan). .

Specifically, Saos-2 cells, which are osteoblast-like cells of Example 5, were dispensed in 96-well plates at a concentration of 1 × 10 ⁴ cells / well, and then BHH10 extracts (0.01, 0,1, 1, 10, 100 μg / ml) was added to 100 μl and incubated for 72 hours. 10 μl of WST-8 (water-soluble tetrazolium salt; 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2,4-disulfophenyl) -2H-tetrazolium, monosodium salt) After the addition, the cells were further incubated for 2 hours, and when WST-8 formazan was formed and developed, the absorbance was measured and measured at 450 nm using an ELISA reader. Cell viability was measured as% of absorbance of the sample treated group relative to the control group.

As a result, as shown in Figure 14 BHH10 extract did not affect the survival rate of Saos-2 cells for 3 days in the range of 0 to 100 ㎍ / ㎖ concentration, it was confirmed that a safe drug without cytotoxicity, 0.01 , Concentrations of 0.1, 1, 10 and 100 μg / ml significantly increased Saos-2 cell survival (FIG. 14).

Experimental Example 9 Measurement of Alkali Phosphatase (ALP) Activity in Osteoblasts of BHH10 Extract

Alkaline phosphatase (ALP), a serum enzyme, is an enzyme that is an indicator of bone formation and is produced during the differentiation of osteoblasts (Zhao Y et al., Biol. Pharm. Bull. , 28 (8), pp1371-1376). , 2005).

ALP activity was measured to determine whether BHH10 extract had a change in osteoblast differentiation. Saos-2 cells, which are osteoblast-like cells prepared and treated as in <Experimental Example 6>, were lysed with 0.1% Triton X-100 (Triton X-100, Sigma, USA). Branson, USA), and centrifuged at 14,000 g, 4 ° C for 20 minutes. The supernatant was incubated with reaction buffer for 3 minutes at 37 ° C., and then the reaction was stopped using 0.1 N NaOH. The absorbance was measured at 405 nm. The standard line was quantified using para-nitrophenol (p-nitrophenol; Sigma-Aldrich Co., MO, USA), and based on this, the ALP amount of the sample was converted.

As a result, as shown in Figure 15, the BHH10 extract of the present invention, after 3 days of treatment on the osteoblasts, it was confirmed that the excellent ALP activity at 0.01 and 0.1 ㎍ / ㎖, BHH10 extract is osteoblast differentiation It can be seen that the promotion (Fig. 15).

Experimental Example 10 Activity Measurement of BHH10 Extract by Alkaline Phosphatase (ALP) Staining in Osteoblasts

Alkaline phosphatase (ALP), a serum enzyme, is an enzyme that is an indicator of bone formation and is produced during the differentiation of osteoblasts (Zhao Y et al., Biol. Pharm. Bull. , 28 (8), pp1371-1376). , 2005).

In order to determine whether BHH10 extract has a change in osteoblast differentiation, ALP activity was measured by staining. Saos-2 cells, which are osteoblast-like cells of Example 5, were dispensed in 12-well plates at a concentration of 1 × 10 ⁵ cells / well, and then BHH10 extracts were prepared by concentration (0). , 0.1, 1, 10 ㎍ / ㎖) was added to 5000 μL and then cultured. The subchondral bone cells prepared by incubating for 14 days were fixed with 70% methanol, treated with staining reagent (BCIP / NBT Liquid substrate system; Sigma-Aldrich Co., MO, USA), shaded, and reacted for 30 minutes at room temperature to confirm the color development. It was.

As a result, as shown in FIG. 16, the BHH10 extract of the present invention showed excellent ALP activity at 0.1 and 10 ㎍ / ml after 14 days of treatment to osteoblasts (a: negative control group, b: BHH10 extract) 0.1 μg / ml treated group, c: 10 μg / ml treated group of BHH10 extract, d: 50 ng / ml treated group of VEGF), and BHH10 extract promoted osteoblast differentiation (FIG. 16).

Experimental Example 11 Measurement of Increased Expression of Vascular Endothelial Growth Factor (VEGF) by BHH10 Extract

Vascular endothelial growth factor (VEGF) is a growth promoter that is synthesized early in the differentiation of osteoblast-like cells, Saos-2 cells, and how BHH10 extract affects the secretion of VEGF. It was confirmed that it was.

Specifically, Saos-2, an osteoblast-like cell line, was cultured in a 48-well plate at a concentration of 5 × 10 ⁴ cells / well, followed by incubation with BHH10 extract in each well. 100 μg / ml) and incubated for 72 hours. To quantify VEGF expression, the cell culture solution was collected and stored at -70 ° C. VEGF quantification was performed using an ELISA kit (VEGF ELISA kit, R & D systems INC., MN, USA). 100 μl of the culture was reacted on a plate of an antibody-labeled kit, and then reacted with a phosphate buffer (PBS) and washed with peroxidase-attached goat anti-mouse immunoglobulin G (R & D). systems INC., MN, USA) and washed again. Color development was carried out using a substrate (TMB; 3,3 ', 5,5'-tetramethylbenzidine) and the reaction was terminated. The absorbance was measured at 450 nm using an ELISA reader.

As a result, as shown in Figure 17, the BHH10 extract of the present invention was confirmed to increase the VEGF secretion, an angiogenesis-promoting factor, in particular BHH10 extract promotes angiogenesis by significantly increasing VEGF secretion even at low concentrations It could be confirmed (Fig. 17).

Experimental Example 12 Measurement of Increased BMP Expression by BHH10 Extract

Bone morphogenic protein-2 (BMP-2) is a protein synthesized at the end of differentiation of osteoblasts. We confirmed how BHH10 extract affects the expression of BMP-2.

Specifically, osteoblasts were cultured in a 48-well plate at a concentration of 5 × 10 ⁴ cells / well, followed by incubation with BHH10 extract in each well (0-100 μg / ml) and incubated for 72 hours. . To quantify the expression level of BMP-2, the cell culture solution was collected and stored at -70 ° C. BMP-2 quantification was performed using an ELISA kit (BMP-2 ELISA kit, R & D systems INC., MN, USA). 100 μl of the culture was reacted on a plate of an antibody-labeled kit, and then reacted with a phosphate buffer (PBS) and washed with peroxidase-attached goat anti-mouse immunoglobulin G (R & D). systems INC., MN, USA) and washed again. Color development was carried out using a substrate (3,3 ', 5,5'-tetramethylbenzidine, TMB) and the reaction was terminated. The absorbance was measured at 450 nm using an ELISA reader.

As a result, as shown in Figure 18, the BHH10 extract of the present invention was confirmed to increase the BMP-2 secretion of bone morphogenic protein, especially at low concentrations significantly increased the BMP-2 secretion BHH10 extract increased the formation Promoting could be confirmed (Fig. 18).

Experimental Example 13 Measurement of Vascular Endothelial Growth Factor (VEGF) and Bone Formation Protein (BMP-2) Gene Expression of BHH10 Extract

VEGF and VEGF in Saos-2 cells, osteoblast-like cell lines treated with BHH10 extract using the reverse transcription polymerase chain reaction (RT-PCR) method of Example 6. The expression level of BMP-2 gene was examined.

As a result, as shown in FIG. 19A, when treated with 1 μg / mL of BHH10 extract, all of the 3, 7, and 14 days increased, and as shown in FIG. 19B, 0, 0.01, 0.1, 1, 10, 100 μg / ml After 14 days of VEGF treatment with BHH10 extract and 50 ng / ml of the positive control, the expression of VEGF gene was increased in a concentration-dependent manner. That is, the BHH10 extract was found to promote osteoblast differentiation (Figs. 19A and 19B).

In addition, as shown in Figure 20, BMP-2 gene expression was shown to increase from 3 days after culture after treatment with 0.1, 1 ㎍ / ㎖ BHH10 extract (Fig. 20).

Therefore, it was confirmed that the BHH10 extract promotes the expression of growth factors and bone morphogenic proteins.

Experimental Example 14 Measurement of BHH10 Extracts Related to Bone Formation Related Gene Expression Promoting Activity

Osteocalcin (OCN), osteopontin (OPN) and collagen type I (type I collagen, Col I) are secreted from osteoblasts, the activity of the OCN, OPN and Col I is osteoblast proliferation Increasing its concentration indicates that bone cell growth and differentiation is active (Collier FM et al ,, Endocrinology, 139 (3), pp1258-1267, 1998).

Therefore, it was examined whether the administration of the BHH10 extract of the present invention affects the activity of OCN, OPN and Col I. Specifically, Saos-2, an osteoblast-like cell line, was cultured in a 48-well plate by dispensing at a concentration of 5 × 10 ⁴ cells / well, followed by 1 μg / ml of whitening extract in each well. Was treated and incubated for 14 days. Cultures were exchanged every 3 days, cultures were collected on day 3 to measure the secretion activity of collagen type I, and RNA was extracted from cells on days 3, 7, and 14 to express OCN, OPN and Col I genes. Was analyzed by RT-PCR.

As a result, as shown in FIG. 21, the expression of OCN, OPN, and Col I increased in all 14 days of culture after treatment with 1 μg / ml BHH10 extract.

In addition, as shown in Figure 22 it was confirmed that the BHH10 extract concentration-dependently promote the activity of the osteogenic differentiation gene (Figs. 21 and 22).

Experimental Example 15 Measurement of Calcium Deposition Promoting Activity of BHH10 Extract

The degree of calcium deposition is a measure of osteocalcification and mineralization (mineralization), the increase in the degree that osteoblast differentiation and bone formation is active.

Thus, it was examined whether the administration of the BHH10 extract of the present invention activates the deposition of calcium. In essence, Saos-2 cells, osteoblast-like cell lines, were seeded in a 24-well plate at a concentration of 1 × 10 ⁴ cells / well to 1% fetal bovine serum (FBS, GIBCO BRL, USA). ) And 1% penicillin / streptomycin (GIBCO BRL, USA), 50 μg / ml ascorbic acid (Sigma, USA) 10 mM β-glycerolphosphate (β-glycerolphosphate, Sigma, USA) After culturing under the added medium conditions, 0.1, 10 μg / ml of the BHH10 extract of the present invention was added to each well and incubated for about 14 days, and the culture solution was exchanged every three days. After incubation, the cells were fixed with methanol, and stained with 0.4% Alinzarin Red S (Sigma, USA) reagent to confirm the degree of calcium deposition. After observation under a microscope (Axiovert 200, ZEISS, Germany), photographs were taken. (200 ×).

As a result, as shown in FIG. 23, intracellular calcium deposition was remarkably observed after 14 days of BHH10 extract treatment (FIG. 23; a: negative control group, b: BHH10 extract 0.1 μg / mL treatment group, c: BHH10 extract 10). ug / mL treatment group, d: VEGF 50 ng / mL treatment group), as shown in Figure 24 was confirmed by quantitatively measuring the intracellular calcium deposition degree dependent on the BHH10 extract concentration (Fig. 24).

Hereinafter, the preparation example containing the BHH10 extract of the present invention will be described, but the present invention is not limited thereto.

Production Example 1 Preparation of Powder

BHH10 Extract 20 mg

Lactose 100 mg

Talc 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Preparation Example 2 Preparation of Tablet

BHH10 Extract 10 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

Production Example 3 Preparation of Capsule

BHH10 Extract 10 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Preparation Example 4 Preparation of Ring

1 g of BHH10 extract

Lactose 1.5 g

Glycerin 1 g

0.5 g of xylitol

After mixing the above components, it was prepared to be 4 g per ring in a conventional manner.

Preparation Example 5 Preparation of Granules

BHH10 Extract 150mg

Soybean Extract 50mg

Glucose 200 mg

600 mg of starch

After mixing the above components, 100 mg of 30% ethanol was added and dried at 60 ° C. to form granules, which were then filled into fabrics.

Preparation Example 6 Preparation of Injection

BHH10 Extract 10 mg

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO ₄ , 12H ₂ O 26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Production Example 7 Preparation of Liquid

BHH10 Extract 20 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

According to the conventional method for preparing a liquid, each component is added and dissolved in purified water, lemon flavor is added, the above components are mixed, purified water is added, the whole is adjusted to 100 ml by adding purified water, and then filled in a brown bottle. The solution is prepared by sterilization.

Production Example 8 Preparation of Healthy Foods

1000 mg of BHH10 extract

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

Vitamin C 10 mg

10 μg biotin

Nicotinic Acid 1.7 mg

50 μg folic acid

Calcium Pantothenate 0.5mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

Production Example 9 Preparation of Healthy Drinks

1000 mg of BHH10 extract

Citric acid 1000 mg

100 g oligosaccharides

Plum concentrate 2 g

1 g of taurine

Add 900 ml of purified water

After mixing the above components according to a conventional healthy beverage production method, and then stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and refrigerated and then stored in the present invention For the preparation of healthy beverage compositions.

Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

As shown above, the mixed herbal medicine extract of Astragalus, Gyeji, and Hwangbaek of the present invention has excellent bone destruction inhibition, bone formation and bone regeneration promoting effect, and there is almost no cytotoxicity, the mixed herbal medicine extract is osteoporosis or bone disease It can be usefully used for the development of preventive and therapeutic agents of dietary supplements and health functional foods.

FIG. 1 is a graph showing the results of column chromatography analysis of the content of formononetin, which is an indicator component of BHH10 (mixed medicinal herb, yellow ginseng and yellowish white mixed herbs) extract.

Figure 2 is a graph showing the results of analyzing the content of the trans-cinamic acid (-trans-cinamic acid), the indicator component of the BHH10 extract.

Figure 3 is a graph showing the results of analyzing the content of berberine (berberine), an indicator component of the BHH10 extract by column chromatography.

Figure 4 is a graph showing the change in animal weight when BHH10 extract administration in ovarian extraction animal model.

Figure 5 is a graph showing the change in the weight of the uterus when the BHH10 extract administration in ovarian extraction animal model.

Figure 6 is a graph showing the change of the small bone area when BHH10 extract administration in ovarian extraction animal model.

Figure 7 is a graph showing the change in bone density when BHH10 extract administration in ovarian extraction animal model.

Figure 8 is a graph showing the change in the growth hormone BHH10 extract administration in ovarian extraction animal model.

9 is a graph showing changes in bone formation factors VEGF and BMP-2 when BHH10 extract is administered in an ovariectomized animal model.

10 is a graph showing the change in bone formation-related factors OCN when BHH10 extract administration in ovarian extraction animal model.

Figure 11 is a figure that suppressed the activity of osteoclasts derived from human subchondral bone tissue when BHH10 extract treatment.

12 is a graph showing the survival rate of osteochondral-derived osteoblasts from human femoral bone when treated with BHH10 extract.

Figure 13 is a graph showing the degree of alkaline phosphatase (ALP) activity in human femoral subchondral osteoblasts osteoblasts treated with BHH10 extract.

14 is a graph showing the survival of osteoblasts (SaOS-2) osteoblasts when BHH10 extract treatment.

15 is a graph showing the degree of alkaline phosphatase (ALP) activity in osteoblasts (SaOS-2) osteoblasts treated with BHH10 extract.

16 is a diagram showing the degree of increase in alkaline phosphatase (ALP) secretion activity in osteoblasts (Saos-2) when BHH10 extract treatment.

Figure 17 is a graph showing the secretion activity of angiogenesis growth factor (VEGF) in osteoblasts (Saos-2) when BHH10 extract treatment.

Figure 18 is a graph showing the increased secretion of bone osteoblastic protein (BMP-2) in osteoblasts (Saos-2) when BHH10 extract treatment.

19 is a diagram showing the expression level of VEGF gene in osteoblasts (Saos-2) when BHH10 extract treatment.

20 is a diagram showing the degree of BMP-2 gene expression in osteoblasts (Saos-2) according to the treatment time when BHH10 extract treatment.

21 is a diagram showing the expression level of BMP-2 gene in osteoblasts (Saos-2) according to the treatment concentration when BHH10 extract treatment.

22 is a diagram showing the gene expression of OCN, OPN and Col I in osteoblasts (Saos-2) according to the treatment time when BHH10 extract treatment.

Figure 23 is a diagram showing the gene expression of OCN, OPN and Col I in osteoblasts (Saos-2) according to the treatment concentration when BHH10 extract treatment.

Figure 24 is a diagram showing the degree of calcium deposition in osteoblasts (Saos-2) when BHH10 extract treatment.

<110> University-Industry Cooperation Group of Kyung Hee University <120> Composition comprising the extract of Astragalus membranaceus          BGE., Cinnamomum cassia and Phellodendron amurensis for          preventing and treating of osteoporesis and bone disease <130> 9p-08-62 <160> 12 <170> KopatentIn 1.71 <210> 1 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> beta-actin forward primer <400> 1 cctggccaag gtcatccatg acaactttgg 30 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> beta-actin reverse primer <400> 2 catgaggtcc accaccctgt tgctgtagcc 30 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> 223 OCN forward primer <400> 3 gaggaccctc tctctgctca ctctgctggc 30 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> 223 OCN reverse primer <400> 4 gtggtgccat agatgcgctt gtaggcgtcc 30 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> OPN forward primer <400> 5 ctcggatgaa tctgacgaat ctcaccattc 30 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> OPN reverse primer <400> 6 cttactctta gggtctagga ctagcttgtc 30 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> coll forward primer <400> 7 tggtcttgga ggaaactttg c 21 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> coll reverse primer <400> 8 agtatctcct ttggcaccat c 21 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> 223 VEGF forward primer <400> 9 tgcacccacg acagaagggg a 21 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> 223 VEGF reverse primer <400> 10 tcaccgcctt ggcttgtcac at 22 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BMP-2 forward primer <400> 11 cagccatgaa tttcacagcc 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BMP-2 reverse primer <400> 12 gggagtttcc atgaagccac 20  

Claims (10)

Pharmaceutical composition for the prevention and treatment of osteoporosis containing the mixed herbal extracts of Astragalus, Gyeji, and Hwangbaek as an active ingredient. The pharmaceutical composition of claim 1, wherein the extract is extracted using water, C ₁ to C ₄ lower alcohols, or a mixture thereof as a solvent. The pharmaceutical composition for preventing and treating osteoporosis according to claim 2, wherein the lower alcohol is ethanol or methanol. According to claim 1, wherein the mixed herbal medicine is a combination composition weight ratio of Astragalus: gyeji: sulfur white is 0.5 to 3: 0.5 to 3: 1 pharmaceutical composition for the prevention and treatment of osteoporosis. [Claim 2] The pharmaceutical composition of claim 1, wherein the mixed herbal medicine has a compounding weight ratio of Astragalus: Gyeji: Yellow and White in a 2: 2: 1 ratio. Pharmaceutical composition for the prevention and treatment of bone diseases, containing the mixed herbal extracts of Astragalus, Gyeji, and Hwangbaek as an active ingredient. The pharmaceutical composition for preventing and treating bone diseases according to claim 6, wherein the bone disease is periodontal disease or bone formation insufficiency. Health food for the prevention and improvement of osteoporosis containing the mixed herbal extracts of Astragalus, Gyeji, and Hwangbaek as an active ingredient. Health food for the prevention and improvement of bone diseases containing the mixed herbal extracts of Astragalus, Gyeji, and Hwangbaek as active ingredients. 10. The method for preventing and improving bone disease of claim 9, wherein the bone disease is periodontal disease or bone formation insufficiency.

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