KR20030031418A - Extract of herbal mixture and pharmaceutical compositions for prevention or treatment of osteoporosis - Google Patents

Abstract

PURPOSE: A pharmaceutical composition containing an extract of mixed natural drugs is provided which has inhibition effect on proliferation of osteoblast and osteoclast is provided . It is effectively used in prevention and treatment of osteoporosis and bone degenerative diseases, and also used as a health supplementary food. CONSTITUTION: The pharmaceutical composition contains a water, alcoholic or aqueous alcoholic extract of mixed drugs selected from the group consisting of (a)Glycyrrhizae Radix, Astragali Radix, Fructus Jujubae, Lonicera japonica Thunb, Fructus Lycii, Cortex Eucommiae and Cynanchi Wilfordi Radix, (b)Glycine Semen Nigra, Rhynchosia nulubilis Semen, Canavalia gladiata DC. and Dolichoris Semen and (c)Cimicifugae Rhizoma, Fructus Arctii, Folium Mori, Radix Puerariae, Radix Bupleuri, Folium Perillae, Herba Agastachis and Cinnamomi Ramulus.

Description

Extract of herbal mixture and pharmaceutical compositions for prevention or treatment of osteoporosis

The present invention relates to a mixed herbal medicine extract, and more particularly, to a mixed herbal medicine extract effective in preventing and treating osteoporosis.

Osteoporosis is apt to be fractured even with a small impact because the bone tissue is thinned due to the reduction of bone tissue and the thinning of the bone, thereby widening the bone marrow cavity, and weakening the bone as the symptoms progress. Bone mass is influenced by several factors, including genetics, nutrition, hormone changes, differences in exercise and lifestyle, and the causes of osteoporosis include old age, lack of exercise, low weight, smoking, low calcium diet, menopause, Ovarian ablation and the like are known. On the other hand, although there are individual differences, blacks have higher bone mass due to lower bone resorption levels than whites, and bone mass is highest at 14-18 years old and decreases by about 1% per year. Especially in women, bone reduction continues after 30 years of age, and when menopause reaches bone growth rapidly due to hormonal changes. In other words, estrogen concentration rapidly decreases at the end of menopause, when a large amount of B-lymphocytes are produced, as in the case of IL-7 (interleukin-7), and the B cell precursor (pre-B) is added to the bone marrow. cells) accumulate and thereby increase the amount of IL-6, thereby increasing the activity of osteoclasts, which eventually leads to a decrease in bone mass.

As described above, osteoporosis is unavoidable for elderly people, especially postmenopausal women, and as the population ages in developed countries, interest in osteoporosis and its therapeutics is gradually increasing. It is also known that there is a market of about $ 130 billion related to the treatment of bone diseases worldwide and is expected to increase further. Therefore, many research institutes and pharmaceutical companies around the world are investing heavily in the development of bone disease treatments. .

Materials currently used to treat osteoporosis include estrogen, androgen anabolic steroids, calcium preparations, phosphates, fluoride preparations, ipriflavones, and vitamin D ₃ . In 1995, Merck, USA, used aminobisphosphonate, and in 1997, Lilly Co., USA, used raloxifene, which acts as a selective estrogen receptor modulator (SERM). It was developed as a treatment.

Meanwhile, conventional osteoporosis therapeutic agents are mostly estrogen-based substances, and estrogen-based substances are known to exhibit side effects such as cancer, gallstones, and thrombosis when administered for a long time. Osteoporosis, however, cannot be treated with short-term administration of the drug, and long-term administration of the drug is essential. Therefore, even when the drug is administered for a long time, there is no side effect as described above and to develop a new substance having an excellent efficacy enough to replace estrogen.

Accordingly, the present inventors have completed the present invention by revealing that the herbal extracts in combination with various herbal medicines can be effectively used to prevent and treat osteoporosis without side effects by showing the activity of proliferating osteoblasts and inhibiting the proliferation of osteoclasts. .

It is an object of the present invention to provide a mixed herbal extract having osteoblast proliferation and osteoclast proliferation inhibitory activity.

It is also an object of the present invention to provide a pharmaceutical composition or health food for preventing or treating osteoporosis comprising the extract as an active ingredient.

1 is a graph showing the change of ALP (alkaline phosphatase) activity and MTT analysis of cell proliferation rate according to the concentration of extract when the mixed herbal medicine extract 1 of the present invention was treated to osteoblast-like cells Saos-2 cells. ,

2 is a graph showing the effect of inhibiting the cell proliferation of osteoclasts according to the concentration of the mixed herbal medicine extract 1 of the present invention,

Figure 3 compared the uterine weight of rats administered the mixed herbal medicine extract 1 of the present invention in the sham (Sham) group, the group administered 17β-estradiol (E2) and control rats and Bonferroni multiple comparison method It's a graph,

4 is a photograph comparing the tibia of the rats to which the mixed herbal medicine extract 1 of the present invention was administered in the tibia and pathological histology of the Sham group, the group to which 17β-estradiol (E2) was administered, and the control rats,

FIG. 5 shows the trabecular bone area (TBA) of the rats to which the mixed herbal medicine extract 1 of the present invention was administered, the sham (Sham) group, the group to which the 17β-estradiol (E2) group was administered, and the small bones of the control rats. Morphometrically analyzed graph.

Figure 6 is a graph measuring the average cattle bone area (× 10 ⁵ μm 2) after administration of the mixed herbal medicine extract of the present invention to SAM P6 mice.

Figure 7 is a graph measuring the total bovine bone area (× 10 ⁵ μm 2) after administering the mixed herbal medicine extract of the present invention to SAM P6 mice.

In order to achieve the above object, the present invention provides a mixed herbal extract with osteoblast proliferation and osteoclast proliferation inhibitory activity.

In addition, the present invention provides a pharmaceutical composition or health food for preventing or treating osteoporosis comprising the extract as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention provides an extract obtained by extracting a mixed herbal medicine composed of one or more selected from the following groups (a) and (b) with water, alcohol or an aqueous alcohol solution.

(a) licorice, dermis, astragalus, jujube, honeysuckle, goji berry, soybean curd, gold and silver squid;

(b) Black beans (or soybeans), weak beans (rats and soybeans), small beans, and white beans.

In the above, the mixed herbal medicine may further comprise one or more of the herbal medicine and / or antler of the following (c) group.

(c) Horse riding, allies, leaflets, mulberry, seahawks, leaflets, wags and cinnamon.

The present invention provides an extract from which all the mixed herbal medicines are arbitrarily combined from each group. In a preferred embodiment of the present invention, 1) licorice, black bean and horseback riding, 2) Astragalus and medicinal soybeans, 3) Astragalus and antler, 4) Astragalus, baekshou, medicinal beans, antler and cinnamon, 5) Astragalus, baekshou, medicinal beans and Cinnamon, 6) Astragalus, medicinal beans and cinnamon, 7) Astragalus, baekshou and medicinal beans, 8) Astragalus, medicinal beans, white migraine and cinnamon; Provided is an extract extracted with alcohol or aqueous alcohol solution.

In addition, in a preferred embodiment of the present invention, 1) 2 parts by weight of licorice, 2 parts by weight of black beans and 1 part by weight of horse riding, 2) 5 parts by weight and 5 parts by weight of soybeans, 3) 5 parts by weight 5 parts by weight of antler and 4 parts by weight of antler, 4 parts by weight of yellow sugar, 4 parts by weight of white sewage, 4 parts by weight of medicinal beans, 1 part by weight of antler, and 0.5 parts by weight of cinnamon 5) 4 parts by weight of yellow sugar, 4 parts by weight of white sugar and 4 parts by weight of soybeans. Part, cinnamon 0.5 part by weight 6) 8 parts by weight of astragalus, 8 parts by weight of soybeans and 1 part by weight of cinnamon, 7) 1 part by weight of astragalus, 1 part by weight of white sewage and 1 part by weight of soybean 8) 8 parts by weight of astragalus, 8 parts by weight of soybean 8 parts by weight of white flakes and 1 part by weight of cinnamon and 9) 8 parts by weight of Astragalus, 8 parts by weight of white sewage, 8 parts by weight of soybeans, 8 parts by weight of white flakes and 1 part by weight of cinnamon.

In the present invention, the mixed herbal medicines are extracted using water, alcohol or alcoholic aqueous solution. The aqueous alcohol solution may be selected from the group consisting of 5 to 100% ethyl alcohol (ethyl alcohol), 5 to 100% methyl alcohol (methyl alcohol) and spirits.

The mixed herbal medicine extract of the present invention can be usefully used for the prevention or treatment of osteoporosis because it has the activity of proliferating osteoblasts and inhibiting the proliferation of osteoclasts.

Licorice, dermis, Astragalus, Jujube, Indong, Gojija, Tochung, Geummihwa and Baekseohsu of the group (a) acts as a decoction medicine or detoxification or energy enhancement and easy to harmonize with other drugs.

Licorice (Lax Radix Glycyrrhizae, scientific name Glycyrrhiza uralensis Fisch. It is written that it promotes stamina, heals wounds, and detoxifies. In addition, Myeong-seol ((名 록) warms the inside, breathless and stuffy, and the internal function is not used smoothly in seawater, stop thirst, the meridians (經脈) to communicate well, and blood (氣血) It is said to be beneficial and detoxifying. In Dongbogam (東 醫 寶 鑑), licorice is used with other herbal medicines to act as a supplement and aid the medicine. Licorice is used as it is, or the skin removed from the roots, and is contained in the Korean Pharmacopoeia, Chinese Pharmacopeia, and Japanese Pharmacy. Licorice is Gancaonin, p-Hydroxybenzoic acid, Scopoletin, Glycyrol, Glycycoumarin, Licocoumarone, Licouma Contains chemical components, such as benzofuran.

Dermis (La Latin name Pericarpium Citri, scientific name Cirtus unshiu MARKOVICH, scientific name Rutaceae) is the first herbal medicine recorded in Neonbonchocho, and used with other Chinese medicines for indigestion, anorexia, vomiting, daughter's disease, phlegm, etc. By harmonizing the effect and increases the biography. Dermal components include d-limonine, hesperidin, myoinositol, vitamin C, and flavonoid glycosides.

Astragalus (Latin name Astragali Radix, scientific name Astragalus membranaceus Bunge, legumes) is used as a representative example, in addition to the immune system to increase the white blood cell count, inhibit aging, cardiac action, and platelet aggregation. Ingredients are known as astragaloside I, II, IV, soyasaponon I, formononetin, palmitic acid, linoleic acid, astraglalan I, II, III.

Jujube (大 라틴; Latin name Fructus Jujubae, scientific name Zizyphus jujuba MILL., Scientific name Rhamnaceae) is the first Chinese herbal medicine recorded in Neonbonbyeongchocho. And prevent the side effects of Chinese medicine. Jujube contains protein, sugars, vitamins A, B ₂ , C, calcium and phosphorus.

Honeysuckle (La Latin name Caulis Lonicerae, scientific name Lonicera japonica THUNB., The scientific name Caprifoliaceae) is the first herbal medicine recorded in the new agricultural herbaceous colony and is used for influenza, infections, boils, numbness and arthritis. Phosphorus components include flavonoids such as linocerin, tannins and alkaloids.

Gugija (Latin name Fructus Lycii, scientific name Lycium chinensis MILL., Scientific name Solanaceae) is the first Chinese herbal medicine recorded in the New Nongbyeon herbaceous medicine, and it is a medicine for the weakness of liver and kidney. It is used for joint pain, dizziness, gloomy eyes, exhausting cough, and sorrow. Ingredients of the wolfberry are 3.39 mg% of carotene, 0.23 mg% of vitamin B1, 0.33 mg% of vitamin B2, 0.33 mg% of vitamin B, 1.7 mg% of nicotinic acid, 3 mg% of vitamin C, and beta-sitosterol and linoleic acid. ) Is contained.

Toothworm (Latin name Cotex Eucommiae, scientific name Eucommia ulmoides OLIV.) use.

Dutong contains 6-10% gutta-percha, 20.7 mg of vitamin C, 0.066 mg of alkaloids, and 6.5 mg of pectin.

Flos Lonicarae, scientific name Lonicera japonica THUNB., The scientific name Caprifoliaceae, is the first Chinese herb to be used in inflammatory, isolated, infectious dysentery, and febrile diseases. Gold and silver components include luteolin, inositol, saponin, tannin, and the like.

Baek Ha Shou (latin Rhizoma Cynanchum) is traditionally used as a substitute for sewage in Korea. It is classified as a drug of Sasang constitution by Dong-Suse Suwon in Yeomma. It is often used in 白 何 烏 寬 中 湯 or Red Baek Haoijungtang. Baeksuo is a nourishing tonic blood pill is used to blacken the head, chronic tingling symptoms, joint pain, decreased sexual function, hemorrhoids, intestinal bleeding, boils are not good. The components of baeksuo include sarcostin, deacylmetaplexigenin, linolein, kidjolanin, caudatin, penupogenin, etc. have.

In addition, the black bean (or soybean), medicinal beans (soybean, seomoktae), small bean and white bean in the group (b) have a function of promoting the function of five intestines and promoting the function of five intestines. .

Black bean (Semen Glycine Nigrae, scientific name Glycine max Merr.名醫 別 錄) to swell the swelling, soreness and swelling and use in the pain and disorders of functional urination, the function of the blood and five intestine (5 차) function to relieve the stiffness and Odu (one of the herbs) Detoxify and roast it to make it powdery and have a lot of heat in it, and it is used for swelling, swelling, abdominal pain and digestion. In the main herbaceous tree (本草綱目), it treats diseases of the kidneys, improves water pressure, lowers the energy, uses it for all kinds of wind fever, activates blood, and detoxifies the drug. I said. He also said that eating edible herbs in the main herbaceous wood gave rise to weight, energy and weakness. In Dongbogam (東 醫 寶 鑑), it harmonizes the inside, lowers the spirit, and communicates with the vein. The drug is beneficial to kidneys and is suitable for kidney disease. The origin of black bean is soybean (black soybean) and is listed in the Korean Herbal Standards. Black bean is mainly composed of chemical components of isoflavones and saponins, and contains trace amounts of genistein and daidzein.

Medicinal beans are also called frosted or rat-eyed beans and have the same potency and ingredients as black bean.

The pea beans (scientific name Canavalia gladiata DC.) Are annual seeds of vines. Treats hiccups, vomiting, abdominal pain, dysentery, whooping cough and sinusitis. Strengthen the spleen. Ingredients include urease, hemagglutinine, and canavanine.

Baekpyeon (白 라틴; Latin name Dolichos Semen, scientific name Dolichoris lablab L., scientific name Leguminosae) is a white, dried seed that is round in egg shape. It has the effect of strengthening the stomach, harmonizing the middle, inhibiting heat and reducing moisture. Treat the loss of appetite caused by vomiting and diarrhea. The taste is sweet and the properties are slightly warm. It contains 0.227% protein, 0.018% fat, 0.57% carbohydrate, calcium, phosphorus, zinc and iron, and has trypsin inhibitor, amylase inhibitor, hemagglutinin A, B is contained.

In addition, horseback riding, ally of the (c) group, upper leaves (mulberry leaves), brown roots, shiho, lobule, kwakyang and cinnamon has the effect of raising the yanggi.

Horseback riding (升麻 Latin name Rhizoma Cimicifugae, scientific name Cimicifuga heracleifolia Kom., Scientific name Ranunculaceae) is the first Chinese herbal medicine to be recorded in the Myeongseol, which includes spinal diseases, parasitic diseases, abdominal pain, epidemic disease, headache, Horseback riding is used for tumors, laryngeal pain and stomatitis. In addition, the herbaceous tree (本草綱目) eliminates the anti-vibration (을) and heals blood (혈) to treat dizziness caused by the lack of good Yang (기), pain in the chest and flanks, frequent diarrhea and after heavy, lob, It is said to be used for uterine bleeding, bloody urine, sexual dysfunction and cold feet. Dong Bogam (東 醫 寶 鑑) said that when used in the lack of energy for the action of horse riding, it acts to boost yang. The horse riding part is underground view and is registered in the Korean and Chinese Pharmacopoeia. Horse riding isoperulic acid, cimigenol, 3-0-β-D-xylopyranosyl siminigenol (3-0-β-D-Xylopyranosyl cimigeniol), acetylsenmagmanol xyllo It contains chemical components such as seeds (Acetylshengmanol xyloside), 25-Anhydrocimigenol, and acerinol.

The allies (牛蒡子; Latin name Fructus Arctii, scientific name Arctium lappa L., scientific name Compositae) are the first herbal medicines recorded in the Myeong-seung Myeongrok. Used for rashes and pain in the throat. Allies contain 25-30% of artin and fatty oils, and artinti produces arctigenin and glucose by hydrolysis.

The upper lobe (La Latin name Folium Mori, scientific name Morus alba L., scientific name Moraceae) is the first herbal medicine recorded in the new agricultural herbaceous botanical herb. use. The upper lobe contains rutin, quercetin, isoquercetin, moracetin, traces of beta-sitosterol, campesterol, and lupeol. , Myoinositol (0.18%), contains chemicals such as inosteroster (inkosterone), Vitamin C 200-300 mg%, Glutathione 140-400 mg, Folic acid 105 ㎍%, Polynin acid (folinin ) 22 μg%, vitamin B1 460 μg%, vitamin B2 300-800 μg%, copper 10 ppm, zinc 16 ppm, boron 35 ppm, manganese 270 ppm.

Brown root (Lax Radix Puerariae, scientific name Pueraria thunbergiana BENTH., Legacy Legminosae) is the first Chinese herbal medicine recorded in Neonbonbyeoncho (으로), cold, thirst, beef, rash, diarrhea, high blood pressure Used for discomfort caused by the back neck. The root roots are puerarin, isolaflavone, puerarin xyloside, daidzein, beta-sitosterol, arachinic acid, Starch and the like.

Siho (柴胡; Latin name Radix Bupleuri, scientific name Bupleurum falcatum L., scientific name Umbelliferae) is the first Chinese herbal medicine recorded in Neonbonchobyeong (農), recurring colds and fever, discomfort in the chest, mouth soreness, It is used for headache, dizziness, malaria, hernia, menstrual relief, uterine sewage and hernia. Seahawks include bupleurumol, oleic acid, linoleic acid, palmitic acid, psychosaponin ABC (saikosaponin A, B, C), and psychogenin FEG (saikogenin F, E) , G) and the like.

The foliar leaf (La Latin Folium Perillae, scientific name Perilla frutescens L., the scientific name Labiatae) was the first Chinese herbal medicine recorded in the new agricultural herbaceous colony (神農 本草 經 集注), cold, chills, fever, cough, chest swelling. It is used to prevent miscarriage of pregnant women and to promote digestion of crabs and fish. The lobules contain perylaldehyde, ι-limonene, alfa-pinene, arginine, cumic acid, and the like.

Gwahyang (Latin Herba Agastache, scientific name Agastache rugosa O. Kuntze) and Gwanghyanghyang (Lamin Herba Pogostemonis, scientific name Pogostemon cablin BENTH.) It is used for nausea, vomiting, boredom, indigestion, vomiting and diarrhea, runny nose and headache. The components of the flavour include more than 80% of methylchavicol and include anethole, anisealdehyde, alpha-limonene and alpha-pinene. The broad fragrance is 52-57% of essential oils with patchouli alcohol and contains eugenol, cinnamic aldehyde and pagostol.

Cinnamomum (La Ramulus Cinnamomi, scientific name Cinnamomum cassia PRESL.) Is the first Chinese herbal medicine to be recorded in Neonbonchobyeong (경), which is a fragile condition, abdominal diarrhea, cold body, joint pain, menstruation. Used for impurities. Cinnamic aldehyde makes up 75-90% of essential oils.

In addition, Cervus Cornu is a representative animal herb, which is defined as the hairy and unosteophilized young horn of Mae Rok ( C. nippon Swinhoe ) or Marok ( C. elaphus Milne-Edwards) and related animals (Deer and Cervidae ). (Korean Pharmacopoeia). Deer antler has the effect of Jang Won-yang Bogi-Bum Ikjeongsu Kang Geungol, strengthening energy and strengthening body and mental power. 효능 精髓) is widely used in clinical practice. In particular, the herbaceous wood is reported to promote regular shortage (精 氣 不足) and satisfy the blood vessels (血虛). Antler Growth Stimulant (AGS) is an effect on the growth of antlers and is known to be closely related to changes in male hormones including testosterone, prolactin and testosterone. , Growth hormone, IGF-I and thyroxine (T ₄ ) are known to have a significant effect on the growth of antler (Han et al., 1994 , pilose Antler, Hanrimwon Publ., Seoul, Korea) .

As a result of examining the effects of the mixed herbal medicine extract of the present invention on the proliferation of osteoblasts, it was confirmed that there is an excellent proliferative effect on the osteoblasts. In addition, the osteoblast proliferation rate is superior to that of NaF, 17β-estradiol (hereinafter abbreviated as “E2”), which is conventionally used as a therapeutic agent for osteoporosis. In addition, since osteoblasts exhibit ALP (alkaline phosphatase) activity, as a result of examining the effects of the mixed herbal medicine extract of the present invention on ALP activity in osteoblasts, the mixed herbal medicine extract of the present invention has excellent ALP activity rate against osteoblasts. The ALP activity rate is higher than that of E2, which is conventionally used as a therapeutic agent for osteoporosis (see FIG. 1 ).

In addition, as a result of examining the effect of the mixed herbal medicine extract of the present invention on the inhibition of the growth of osteoclasts (osteoclase), the mixed herbal medicine extract of the present invention was found to have a cell growth inhibition effect of osteoclasts (see FIG. 2 ). Therefore, the mixed herbal extract of the present invention was found to increase osteoblast activity and simultaneously inhibit osteoclast activity, thus confirming that it is an ideal agent for preventing and treating osteoporosis.

Animal experiments on osteoporosis were performed in the mixed herbal extract of the present invention in SD (Sprague-Dawley) rats in which type I osteoporosis occurs after menopause. The sham group compared the control group with ovarian extraction as the control group compared with the control group without the ovarian extraction and drug administration. One week after the ovary was removed, the Siamese group and the control group were orally administered drinking water, the E2 group was intraperitoneally injected with 17β-estradiol, and the herbal group was orally administered the mixed herbal extract of the present invention, and the weight of each experimental animal was measured. The results were measured weekly and the tissue weight was measured after a certain period of time, and compared with the weight and tissue weight of the sham group and all the ovaries, except for ovarian extraction, and the control group without the drug administration. While the increase in body weight was significantly lower than the control group during the drug administration period, it can be seen that in the experimental group administered the mixed herbal medicine extract of the present invention, the body weight was increased in almost the same manner as the control group (see Table 7 ). The above results indicate that in the ovary extraction state, the weight rapidly increases with increasing age, and the administration of the mixed herbal medicine extract of the present invention does not have any effect on the increase in body weight with aging after ovarian extraction. In addition, the weight of all tissues except the uterus in the experimental animals of each group did not show any difference, but the weight of the uterus was increased compared to the control group in the Siamese group and the E2 group, while the mixed herb extract group was almost similar to the control group ( FIG. 3 ) . Reference). Since no pathological differences were observed between bone tissues in the experimental animals of each group, it was determined that there was no in vivo toxicity due to administration of the test drug (see FIG. 4 ). In addition, the control group from which the ovary was extracted was reduced by about 50% in the area of the subcetabular bone compared to the normal state (Siamese group), indicating that osteoporosis was induced (see FIG. 5 ). The osseous bone area due to ovarian extraction was increased due to the administration of E2 or the mixed herbal extract of the present invention, which was lower than the normal group, but significantly increased compared to the control group. Administration of the mixed herbal medicine extract of the present invention increased the area of the cattle bone compared to the control group (see FIGS. 5 , 6 and 7 ). Therefore, the mixed herbal extract of the present invention can be effectively used for the prevention or treatment of osteoporosis.

The present invention also provides a pharmaceutical composition for preventing or treating osteoporosis containing the extract as an active ingredient.

The pharmaceutical composition for preventing or treating osteoporosis of the present invention contains the mixed herbal extract as an active ingredient. The mixed herbal extracts can be administered orally or parenterally during clinical administration and can be used in the form of general pharmaceutical formulations. That is, the mixed herbal extract of the present invention may be administered in various oral and parenteral formulations during actual clinical administration, and when formulated, diluents such as fillers, extenders, binders, humectants, disintegrating agents, and surfactants are usually used. Or using excipients. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and such solid preparations may contain at least one excipient such as starch, calcium carbonate, sucrose, lactose and gelatin, etc., in a mixed herbal extract. Are mixed to prepare. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral administration include suspensions, solutions, 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. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. As the non-aqueous solvent and the suspension solvent, 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, glycerol and gelatin may be used.

Dosage units may contain, for example, one, two, three or four times the individual dosage, or they may contain 1/2, 1/3 or 1/4 times. The individual dosage preferably contains an amount in which the effective drug is administered at one time, which usually corresponds to all, 1/2, 1/3 or 1/4 times the daily dose.

The effective dose of the mixed herbal medicine extract is 10 to 1,000 mg / kg, preferably 30 to 3,000 mg / kg, and may be administered 1-6 times a day.

Toxicity test of oral administration, intraperitoneal administration, and subcutaneous injection of the mixed herbal medicine extract of the present invention to rats showed that 50% lethal dose (LD ₅₀ ) by oral administration toxicity test was at least 10 g / kg of water extract. It turned out to be a safe substance.

In addition, the present invention provides a health food for preventing or treating osteoporosis comprising the extract as an active ingredient.

Examples of health foods containing mixed herbal medicine extracts include health foods and favorite products such as juice, tea, jelly, juice, etc., which are mainly composed of mixed herbal medicines, and folk remedies for the purpose of blood supplements, nourishing agents, anti-aging agents, and musculo-enhancers. Etc. can be mentioned.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Preparation of Mixed Herbal Medicine Extract 1

Horse riding, licorice and black bean were mixed in a 1: 2: 2 weight ratio, and 1.5 kg of the powder of the mixed herbal medicines was added to 3 L of a 70% methanol aqueous solution and stirred at room temperature for 2 days. After 2 days, the solution was filtered with filter paper and the process was repeated three times. The filtrate was concentrated under reduced pressure to remove methanol, and then freeze-dried to remove moisture to obtain a mixed herbal extract.

<Example 2-Example 9> Preparation of the mixed herbal medicine extract 2-9

The present inventors prepared the mixed herbal medicine extracts 2 to 9 in the same manner as in Example 1 by mixing the herbal medicine as follows ( Table 1 ).

Herbal medicine Weight ratio Extract 2 Astragalus + Medicinal Beans 1: 1 Extract 3 Astragalus + Antler 1: 1 Extract 4 Astragalus + Medicinal Beans + White Sewage + Cinnamon + Deer Antler 4: 4: 4: 0.5: 1 Extract 5 Astragalus + medicinal beans + white sewage + cinnamon 4: 4: 4: 0.5 Extract 6 Astragalus + Yak + Cinnamon 8: 8: 1 Extract 7 Astragalus + Yak bean + Baek Ha Shou 1: 1: 1 Extract 8 Astragalus + Medicinal Beans + White Bean + Cinnamon 8: 8: 8: 1 Extract 9 Astragalus + Medicinal Beans + Baekpyeon + Baekshou + Cinnamon 8: 8: 8: 8: 1

Experimental Example 1 Osteoblast Proliferation Analysis

The present inventors carried out the following experiment to determine the effect of the mixed herbal extracts prepared in Examples 1 to 9 on the proliferation of osteoblasts (osteoblast).

<1-1> Osteoblast Screening and Cell Culture

The MG-63 cell line and Saos-2 cell line, which have similar properties to osteoblasts, the constituents of bone, were distributed from the Bank of Korea Cell Line at the Cancer Research Institute of Seoul National University.

MG-63 cells were used DMEM medium (Gibco BRL, USA) containing 10% FBS, 100 units / ml penicillin, 100 μg / ml streptomycin, Saos-2 cells 100 units / ml 10% FBS, penicillin , RPMI 1640 medium (100 g / ml) containing streptomycin (Gibco BRL, USA) was incubated in a 5% CO ₂ incubator at 37 ℃ in wet conditions. Medium was changed 2-3 times a week and subcultured once a week.

<1-2> Cell proliferation experiment according to the concentration of the extract: MTT experiment

Saos-2 cell line or MG-63 cell line cultured in Experimental Example <1-1> was inoculated into a 96-well plate, and the mixed herbal medicine extracts 1 to 9 of the present invention were dried to 2 to 2 x 10 ^{-7 per} drug. Six wells were added at each concentration to give a concentration of mg / ml. As a control, no extract was used, and as a comparative group in the Saos-2 cell line or the MG-63 cell line, NaF, 17β-estradiol (hereinafter abbreviated as “E2”), which is currently mainly used as a therapeutic agent for osteoporosis, and 1,25 (OH) ₂ D ₃ and the like were used.

The cell line was incubated for 3 days in a 37 ° C. incubator, and MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide; Triazolyl Blue) was added at a concentration of 0.05 mg / ml. Incubated further 4 hours under conditions. The resulting formazan crystals were dissolved in DMSO and the absorbance at 550 nm was measured with an ELISA reader.

Cell proliferation (%) was calculated as the ratio (%) of absorbance of extract added wells to absorbance of control wells without extract ( Table 2; Saos cells , Table 3; MG-63 cells).

As a result, the mixed herbal medicine extract of the present invention showed an excellent proliferation rate for osteoblast-like cells such as Saos-2 cells, and compared to NaF, E2, and 1,25 (OH) ₂ D ₃ , which are conventionally used as therapeutic agents for osteoporosis. Cell proliferation was better ( Table 2 and FIG. 1 ). In addition, the mixed herbal medicine extract of the present invention showed excellent proliferation rate for osteoblast-like cells such as MG-63 cells ( Table 3 ). Therefore, it was confirmed that the herbal extract of the present invention can be usefully used for drugs and health foods related to the treatment or prevention of osteoporosis.

Concentration (mg / ml) 1 × 10 ^-9 1 × 10 ^-8 1 × 10 ^-7 1 × 10 ^-6 1 × 10 ^-5 1 × 10 ^-4 1 × 10 ^-3 1 × 10 ^-2 NaF - - 113.52 112.62 92.02 102.93 103.56 - E2 - - 103.17 118.11 93.79 96.48 100.07 - 1,25 (OH) ₂ D ₃ - - 93.58 96.01 101.55 103.93 120.88 - Concentration (mg / ml) 2 × 10 ^-7 2 × 10 ^-6 2 × 10 ^-5 2 × 10 ^-4 2 × 10 ^-3 2 × 10 ^-2 2 × 10 ^-1 2 × 10 ⁰ Extract 1 78.53 86.06 89.41 78.37 94.04 78.59 96.63 131.24 Extract 2 97.49 109.56 115.90 129.03 135.92 136.11 117.44 88.97 Extract 3 111.67 116.74 128.77 115.09 140.03 179.66 150.54 117.96 Extract 4 69.65 111.80 107.57 123.36 123.94 88.63 124.33 73.20 Extract 5 95.02 100.99 98.84 120.18 119.50 91.98 114.12 86.78 Extract 6 106.20 101.94 105.58 131.87 109.60 138.79 82.91 80.89 Extract 7 95.39 102.91 96.38 87.68 120.59 144.50 132.77 101.39 Extract 8 103.09 107.90 145.41 120.38 113.91 109.92 91.23 69.80 Extract 9 121.60 121.06 174.76 162.61 132.83 138.02 124.74 84.46

Concentration (mg / ml) 1 × 10 ^-9 1 × 10 ^-8 1 × 10 ^-7 1 × 10 ^-6 1 × 10 ^-5 1 × 10 ^-4 1 × 10 ^-3 1 × 10 ^-2 NaF - - 94.54 85.00 87.22 108.64 137.93 - E2 - - 98.69 108.07 112.31 113.98 116.57 - 1,25 (OH) ₂ D ₃ - - 117.14 126.83 136.50 116.91 85.88 - Concentration (mg / ml) 2 × 10 ^-7 2 × 10 ^-6 2 × 10 ^-5 2 × 10 ^-4 2 × 10 ^-3 2 × 10 ^-2 2 × 10 ^-1 2 × 10 ⁰ Extract 1 127.14 113.11 110.01 118.01 125.85 114.25 85.77 120.43 Extract 2 100.47 119.41 113.67 120.75 133.82 125.33 107.04 124.05 Extract 3 112.80 129.58 103.40 127.81 139.73 141.09 130.70 126.65 Extract 4 109.24 117.26 146.94 133.98 156.03 144.65 110.78 168.72 Extract 5 145.56 129.92 138.94 146.55 124.53 128.38 140.46 160.52 Extract 6 96.39 109.12 116.15 108.79 106.09 112.26 91.62 141.41 Extract 7 108.32 110.87 114.97 112.98 125.75 115.29 148.21 162.92 Extract 8 111.81 93.20 101.10 114.64 84.84 125.16 96.88 114.39 Extract 9 124.53 114.85 100.87 102.03 103.01 94.30 141.41 113.04

<1-3> Activity Detection of ALP (alkaline phosphatase)

Since osteoblasts exhibit ALP (alkaline phosphatase) activity, the mixed herbal extract of the present invention was examined by the following method to affect the ALP activity in osteoblasts.

Saos-2 cell lines of the same cell number as in the MTT experiment were treated with the same concentrations and harvested after 3 days of incubation under the same conditions. At this time, NaF, E2, 1,25 (OH) ₂ D _3, etc. were used as a comparison group. Meanwhile, ALP activity was measured using a change in absorbance at 405 nm by decomposing p-nitrophenylphosphate to p-nitrophenol and phosphate. .

As a result, the mixed herbal medicine extract of the present invention showed excellent ALP activity against Saos-2 cells ( Table 4 and Figure 1 ). Therefore, the mixed herbal medicine extract of the present invention can promote bone production by increasing cell activity of osteoblasts as well as cell activity, and thus can be usefully used for drugs and health foods related to the treatment or prevention of osteoporosis.

Concentration (mg / ml) 1 × 10 ^-9 1 × 10 ^-8 1 × 10 ^-7 1 × 10 ^-6 1 × 10 ^-5 1 × 10 ^-4 1 × 10 ^-3 1 × 10 ^-2 NaF - - 94.81 94.56 93.02 87.17 87.05 - E2 - - 96.18 100.92 98.71 90.85 91.03 - 1,25 (OH) ₂ D ₃ - - 192.15 93.92 99.51 182.73 167.46 - Concentration (mg / ml) 2 × 10 ^-7 2 × 10 ^-6 2 × 10 ^-5 2 × 10 ^-4 2 × 10 ^-3 2 × 10 ^-2 2 × 10 ^-1 2 × 10 ⁰ Extract 1 109.57 113.71 112.96 109.03 118.07 114.13 113.92 78.86 Extract 2 104.2 106.11 94.22 105.68 108.53 106.63 101.92 146.21 Extract 3 102.08 94.82 97.95 102.41 97.00 116.11 106.71 114.43 Extract 4 93.10 94.12 87.48 84.41 99.78 103.22 103.80 48.03 Extract 5 97.43 91.76 81.98 79.10 80.16 93.46 84.06 33.60 Extract 6 127.34 117.30 138.72 121.54 119.08 112.54 109.33 111.78 Extract 7 85.53 65.64 76.62 70.95 84.88 107.87 85.59 125.76 Extract 8 103.13 88.25 84.46 80.70 85.35 105.79 96.76 127.68 Extract 9 95.56 117.11 92.65 93.59 80.56 90.36 107.52 139.16

Experimental Example 2 Inhibition of Cell Proliferation of Osteoclasts

In order to examine the effects of the mixed herbal medicine extract of the present invention on the inhibition of the growth of osteoclasts, the present inventors co-cultured the bone marrow cells (mouse bone marrow cells) and ST-2 cells (ST-2 cells) ) To obtain the osteoclasts and performed the following experiment.

<2-1> Coculture of Bone Marrow Cells and ST-2 Cells

<2-1-1> Culture of Bone Marrow Cells

Muscle tissues around femur and bitia of ICR mice (Korean experimental animals, Chungbuk, Negative) were removed cleanly and separated from femur and tibia. Transfer it to a clean bench and place α-MEM (α-minimum essential medium, Gibco BRL, USA) medium containing 10% FBS, 100 units / ml penicillin and 100 μg / ml streptomycin previously iced. Into the femur and tibia respectively. The ends of the femur and tibia were cut and the medium was washed down until the bone marrow was completely drained. Pellets were obtained by centrifugation, which were resuspended in the culture medium, aliquoted into culture flasks, and then cultured in a 37%, wet 5% CO ₂ incubator. Non-tacky cells were collected and resuspended in pronase solution and reacted at 37 ° C. After centrifugation, the precipitated cells were suspended in the culture medium and used for co-culture with ST-2 cells.

<2-1-2> Culture of ST-2 Cells

ST-2 cells were obtained from RIKEN Cell Bank (Tsukuba, Japan). ST-2 cells were cultured using a 5% CO ₂ incubator in wet conditions at 37 ° C. using RPMI 1640 medium containing 10% FBS, 100 units / ml penicillin, 100 μg / ml streptomycin. The medium was changed 2-3 times a week and subcultured once a week. Since ST-2 cells grow in a monolayer on culture plaques, the monolayer was stripped off with 0.25% trypsin solution during subculture.

<2-1-3> Coculture of Bone Marrow Cells and ST-2 Cells

ST-2 cells were treated with trypsin and suspended in α-MEM medium containing 10% FBS. The ST-2 cell suspension was added to a 96-well plate at 5 × 10 ⁴ cells / cm 2 and incubated for 24 hours. Non-adherent bone marrow cells were added to 96-well plates containing ST-2 cells at 5 × 10 ⁵ cell numbers / cm 2. As co-culture medium, a medium of? -MEM containing 10% FBS, 10 ⁻⁸ M 1,25 (OH) ₂ D ₃ , and 10 ⁻⁷ M dexamethasone (dexamethasone) was used. The medium was changed twice a week.

<2-2> Cell proliferation inhibition experiment of osteoclast

On the 7th day of co-culture of bone marrow cells and ST-2 cells, the extracts 1 to 9 were treated, followed by counting the number of multinucleated cells by TRAP (tartrate resistant acid phosphatase) staining. Staining was done using a commercially available kit (Sigma, USA), and TRAP-positive cells (TRAP-positive MNC) with three or more nuclei were measured in osteoclasts and expressed in% relative to control cells.

As a result, the mixed herbal medicine extract of the present invention was shown to have a cell proliferation inhibitory effect of osteoclasts ( Table 5 and Figure 2 ).

Concentration (mg / ml) 2 × 10 ^-4 2 × 10 ^-3 2 × 10 ^-2 2 × 10 ^-1 2 × 10 ⁰ Extract 1 84.42 91.40 41.86 65.58 - Extract 2 95.90 - 62.77 - 45.75 Extract 3 100.10 100.75 87.35 88.00 - Extract 4 97.4 137.9 151.8 122.6 62.9 Extract 5 135.0 144.9 138.5 100.0 68.0 Extract 6 73.9 89.6 56.8 50.7 48.6 Extract 7 98.52 106.39 92.73 89.10 - Extract 8 97.41 107.9 91.83 87.56 72.9 Extract 9 120.72 94.71 98.32 88.93 82.46

In addition, the present inventors treated the 5% sodium hypochlorite solution for 5 minutes after TRAP analysis in osteoclast growth inhibition experiment to remove the cells attached to the plate. In order to measure the degree of loss of bone tissue in the plate using an image analyzer, the area of the pit was measured and expressed as% of the pit area of the sample addition group to the pit area of the control well.

As a result, the mixed herbal medicine extract of the present invention was shown to have a cell proliferation inhibitory effect of osteoclasts ( Table 6 ). That is, the mixed herbal medicine extract of the present invention increases the activity of osteoblasts and at the same time inhibits the activity of osteoclasts, which is considered to be an ideal agent for the prevention and treatment of osteoporosis.

Concentration (mg / ml) 2 × 10 ^-4 2 × 10 ^-3 2 × 10 ^-2 2 × 10 ^-1 2 × 10 ⁰ Extract 1 87.3 95.1 78.4 72.5 - Extract 2 94.0 - 63.6 - 52.8 Extract 3 93.9 98.2 71.8 84.0 - Extract 4 69.4 117.6 67.9 99.1 77.8 Extract 5 141.6 91.3 113.1 74.5 39.9 Extract 6 80.3 102.9 81.9 51.7 47.2 Extract 7 101.7 99.5 104.0 97.3 - Extract 8 96.3 100.2 89.3 86.7 78.0 Extract 9 117.1 97.9 93.2 83.9 76.4

Experimental Example 3 Animal Experiments on Osteoporosis Model Animal Experiments on Ovariectomized Rats

The present inventors conducted ovarian extraction experiments on females of SD (Sprague-Dawley) rats who developed type I osteoporosis after menopause to study animal osteoporosis, and examined SAM P6 mice having type II osteoporosis. The change in the area of the subcetabular bone was measured.

<3-1> Ovarian Extraction and Drug Administration

Sprague Dawley rats of about 200-300 g body weight of 10 weeks old were used as experimental materials. Ovarian extraction was performed in all female rats of the control group and the test group except the sham group (normal group). Female rats were anaesthetized by intramuscular injection of 5 mg / 100 g of ketamine (Ketamine; Yuhan, Korea) and 1 mg / 100 g of xylazine (Bayer Korea, Korea) into the left and right hind femur of the rat. The lower abdomen was dissected around the midline and the left and right ovaries were extracted. 0.3 ml of antibiotic (Sulpaporte-4, Unichem Co., Ltd.) was injected intraperitoneally to prevent infection, and the peritoneum, abs and skin were sutured with silk and nylon yarns.

Sham (sham) group is a comparison group to the control group without the ovary extraction and drug administration to all surgically operated animals except ovary extraction exists to compare the changes due to ovarian extraction with the control group. The control group, on the other hand, is for comparing the changes due to drug administration compared to animals of the administration groups that underwent ovarian extraction and drug administration. One week after the ovary was removed, the Siamese group and the control group were orally administered drinking water, and the E2 group was intraperitoneally injected with 17β-estradiol at 1 μg / kg / day. In addition, the extracts 1 to 9 of the present invention were orally administered at 5 g / kg / day for 9 weeks, and genistein, a component of the medicinal beans, was intraperitoneally injected at 10 mg / kg / day for 9 weeks.

<3-2> Measurement of changes in body weight and tissue weight by drug administration

After the ovary was extracted by the above method, the weight of the experimental animals to which the drug was administered was measured every week, and the tissue weight was measured after a certain period of time to extract the siamese group and ovaries which performed all operations except ovarian extraction. Boneferroni multiple comparison method) was used to compare the body weight and tissue weight of the control group.

As a result, there was no change in body weight or uterine weight when Genistein was administered compared with the control group. In other words, it is thought that there is no hormonal change that is large enough to indicate a change in body weight or uterine weight, whereas the E2 group shows uterine hypertrophy as a side effect, whereas the extract of the present invention is considered as a safe drug because the above symptoms do not appear. Table 7 , Table 8 , Figure 3 ).

Changes in Weight of Experiment Animals by Drug Administration Week Control Siamese E2 Extract 1 10 237.49 ± 7.41 233.29 ± 5.71 244.97 ± 8.06 256.75 ± 8.32 11 256.74 ± 6.10 242.99 ± 5.35 262.41 ± 6.91 256.65 ± 7.13 12 285.14 ± 6.03 ^** 251.69 ± 5.52 ^# 272.33 ± 7.13 281.93 ± 8.50 ^* 13 310.79 ± 5.94 ^** 260.17 ± 5.64 ^## 281.00 ± 6.30 308.76 ± 9.36 ^** 14 326.45 ± 7.09 ^** 270.05 ± 6.76 ^{** ##} 293.05 ± 7.74 ^** 324.62 ± 10.38 ^** 15 336.18 ± 7.97 ^** 275.66 ± 5.70 ^{** ##} 302.64 ± 8.78 ^** 330.18 ± 12.19 ^** 16 345.38 ± 7.95 ^** 278.34 ± 6.46 ^{** ##} 299.82 ± 7.76 ^** 339.76 ± 12.60 ^** 17 343.13 ± 8.12 ^** 282.68 ± 7.31 ^{** ##} 306.27 ± 8.47 ^** 342.91 ± 11.95 ^** 18 357.26 ± 8.58 ^** 285.88 ± 6.72 ^{** ##} 308.97 ± 8.56 ^{** #} 346.71 ± 11.77 ^** 19 364.77 ± 8.59 ^** 292.07 ± 6.83 ^{** ##} 319.14 ± 9.06 ^** 348.82 ± 11.34 ^** 20 371.21 ± 8.45 ^** 300.32 ± 6.95 ^{** ##} 327.96 ± 8.22 ^** 356.27 ± 11.87 ^**

* As compared to 10 weeks of age; p <0.05, **; p <0.01,

# Compared to control; p <0.05, ##; p <0.01

tibia Lumbar vertebra Control 100.0 ± 7.6 100.0 ± 3.4 Siamese 247.1 ± 15.3 ** 138.8 ± 7.8 ** E2 group 148.5 ± 6.6 * 132.0 ± 4.2 ** Genistein group 137.6 ± 6.0 * 128.2 ± 4.4 * Extract 1 137.2 ± 8.3 * 100.9 ± 3.8 Extract 2 134.7 ± 7.9 126.0 ± 6.8 Extract 3 132.5 ± 10.0 120.9 ± 4.0 Extract 4 147.5 ± 4.9 ** 131.4 ± 4.4 ** Extract 5 142.1 ± 6.2 * 128.0 ± 4.8 * Extract 6 133.9 ± 7.2 121.8 ± 5.0 Extract 7 132.0 ± 4.8 124.4 ± 4.2 Extract 8 139.4 ± 6.6 * 128.8 ± 2.6 ** Extract 9 140.8 ± 7.2 * 130.0 ± 3.8 **

* As compared to 10 weeks of age; p <0.05, **; p <0.01

<3-3> histopathological observation

The collected femoral tissue was fixed in 10% formalin solution and demineralized in formic acid. Observation sites of bone tissue were cut with a surgical knife, followed by a dehydration process ranging from 70 to 100% alcohol and acetone. Paraffin-embedded bone tissue was cut to 5 μm with a microtome, stained with hematoxylin and eosin (H & E), and observed under an optical microscope.

As a result, no pathological difference was observed between the bone tissues of each group. Therefore, it was judged that there was no toxicity in vivo due to the administration of test drugs. In the administration group of the mixed herbal medicine extract it was confirmed that there is a lot of shochu bone ( Fig. 4 ).

<3-4> Morphometric Analysis

Morphometric analysis was performed in the lumbar and tibia of each group by the following method. Quantitative image analysis system (Wild Leitz Co.) digitizer (digitizer) of the lumbar and tibia in each area of the soju was automatically calculated to calculate the area of trabecula (trabecula). In the proximal part of each tibia, the average length of the subcetabular bone in the inside of the rectangle having a reference area of 2 × 10 ⁶ µm ² , the length of the horizontal side of the lower part of the growth plate about 2/3 of the growth plate length, After calculating the number of bone fragrances inside the rectangle, the average area was multiplied by the number to determine the area of the subcetabular bone of each bone sample, and then statistically processed.

As a result, the control group extracted from the ovary was reduced by about 50% of the area of the subcetabular bone compared to the normal state (Siamese group), indicating that osteoporosis was induced. In addition, the area of osseous bone due to ovarian extraction was increased due to the administration of E2 or mixed herbal extracts, especially extract 1, which was lower than that of the Siamese group (P <0.01) but significantly increased compared to the control group (P <0.05). ). Administration of the mixed herbal medicine extract of the present invention increased the area of the minor bone by about 40% compared to the control ( Fig. 5 ). Therefore, it can be seen that the mixed herbal extract of the present invention can be effectively used for the prevention and treatment of osteoporosis.

<3-5> Suzhou bone area experiment

The present inventors have described SAM P6 in which type II osteoporosis occurs by reference to a reference on the effect of Chinese medicine such as Kim Sook-Sook on bone loss in ovarian-depleted rats and SAM P6 mice (Kim Jung-Sook et al., 1998, 42, 220-228) After the administration of the extract of the present invention in mice was measured the change in the area of the small bone. Specifically, the extracts 1 to 9 of the present invention were orally administered at 5 g / kg / day, and the control group was administered under the condition of not administering anything, and then measured the area of the small bone.

As a result, the group administered with the extract for 14 days increased the mean sub-catheteral bone area ( FIG. 6 ) and the total sub-catheter bone area ( FIG. 7 ) compared to before the administration of the extract for 30 days. Compared with the mean minor bone area was increased ( FIG. 6 ), the total minor bone area was also increased with respect to the control group at 30 days ( FIG. 7 ).

Experimental Example 4 Acute Toxicity in Rats

Acute toxicity test was performed using 6-week-old specific pathogen-free (SPF) SD rats. Two animals per group were suspended orally administered once with a dose of 1 g / kg / ml in suspension of the mixed herbal medicine of the present invention in 0.5% methylcellulose solution, respectively. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed. Hematological and hematological examinations were performed. Necropsy was performed to observe abdominal and thoracic organ abnormalities.

As a result, no significant clinical symptoms or dead animals were noted in all animals treated with the test substance, and no toxic changes were observed in weight changes, blood tests, blood biochemical tests, and autopsy findings. Therefore, the mixed herbal medicine extracts of the present invention do not show a toxic change even up to 10 g extract / kg in rats and the minimum lethal dose (LD ₅₀ ) was determined to be a safe substance of more than 10 g / kg extract.

As described above, since the mixed herbal medicine extract of the present invention has an activity of proliferating osteoblasts and inhibiting the proliferation of osteoclasts, osteoporosis, degenerative bone disease and bone diseases such as rheumatoid arthritis, etc. It can be usefully used for prevention or treatment. In addition, since it is not toxic at all, it can be widely used as a dietary supplement.

Claims (14)

The extract of the mixed herbal medicines mixed with one or more selected from the following groups (a) and (b), respectively, with water, alcohol or an aqueous alcohol solution. (a) licorice, dermis, astragalus, jujube, honeysuckle, goji berry, soybean curd, gold and silver squid; (b) Black bean (or soybean), weak bean (rat bean, Seomoktae), small bean and white bean The extract according to claim 1, wherein the mixed herbal medicine further comprises one or more herbal medicines and / or antlers of the following group (c). (c) horseback riding, allies, leaflets (mulberry leaves), brown roots, seahawks, leaflets, wagyang and cinnamon The extract according to claim 1 or 2, wherein the extract is mixed with 2 parts by weight of licorice, 2 parts by weight of black bean, and 1 part by weight of horse riding. The extract according to claim 1 or 2, wherein the extract is mixed with 5 parts by weight of Astragalus and 5 parts by weight of medicinal beans. The extract according to claim 1 or 2, wherein the extract is mixed with 5 parts by weight of Astragalus and 5 parts by weight of antler. The extract according to claim 1 or 2, wherein the extract is mixed with 4 parts by weight of sulfuric acid, 4 parts by weight of white sewage, 4 parts by weight of medicinal beans, 1 part by weight of antler, and 0.5 parts by weight of cinnamon. The extract according to claim 1 or 2, wherein 4 parts by weight of Astragalus, 4 parts by weight of white sewage and 4 parts by weight of soybeans, 0.5 parts by weight of cinnamon are mixed. The extract according to claim 1 or 2, wherein the extract is mixed with 8 parts by weight of Astragalus, 8 parts by weight of medicinal beans and 1 part by weight of cinnamon. The extract according to claim 1 or 2, wherein the extract is mixed with 1 part by weight of Astragalus, 1 part by weight of white sewage and 1 part by weight of medicinal beans. The extract according to claim 1 or 2, wherein the extract is mixed with 8 parts by weight of Astragalus, 8 parts by weight of medicinal beans, 8 parts by weight of white bean, and 1 part by weight of cinnamon. The extract according to claim 1 or 2, wherein the extract is mixed with 8 parts by weight of Astragalus, 8 parts by weight of white sewage, 8 parts by weight of medicinal beans, 8 parts by weight of white yeast, and 1 part by weight of cinnamon. The extract according to claim 1, wherein the aqueous alcohol solution is selected from the group consisting of 5 to 100% ethyl alcohol, 5 to 100% methyl alcohol and spirits. A pharmaceutical composition for preventing or treating osteoporosis, comprising the extract of claim 1 as an active ingredient. Health food for the prevention or treatment of osteoporosis containing the extract of claim 1 as an active ingredient.