KR100799492B1 - A herbal mixture extract of Rehmanniae Radix Preparata and Drynaria fortunei Kze. J. sm. and composition comprising the same for prevention and treatment of osteoporosis - Google Patents

Abstract

The present invention relates to a mixed herbal extract of Sukji sulfur and bone chain beam and a composition for preventing and treating osteoporosis using the same as an active ingredient.

The mixed herbal medicine extract of the present invention effectively inhibits the formation and activity of osteoclasts, and thus can be widely used as medicines and health foods useful for the prevention and treatment of osteoporosis.

Description

A herbal mixture extract of Rehmanniae Radix Preparata and Drynaria fortunei (Kze.) J. sm. and composition comprising the same for prevention and treatment of osteoporosis}             

1 is a diagram showing the inhibition of osteoclast proliferation by the mixed herbal medicine extract (Sukjihwang water extract fraction (0.15 ~ 4.9μl / ㎖) and bone chain extract extract fraction (0.05 ~ 4.0μl / ㎖) of the present invention.

Figure 2 is a diagram showing the inhibition of osteoclast proliferation by the mixed herbal medicine extract (Sukjihwang butanol extract fraction (0.5 ~ 14.7μl / ㎖) and bone chain extract extract fraction (0.05 ~ 4.0μl / ㎖) of the present invention.

Figure 3 is a diagram showing the inhibition of osteoclast proliferation by the mixed herbal medicine extract (Sukjihwang ethyl acetate extract fraction (0.25 ~ 6.67 µl / ml) and bone chain extract extraction fraction (0.05 ~ 4.0 µl / ml) of the present invention) .

Figure 4 is a diagram showing the inhibition of osteoclast proliferation by the mixed herbal medicine extract (Sukjihwang ethanol extract fraction (0.625 ~ 5.0µl / ㎖) and bone chain extract extraction fraction (0.25 ~ 2.0µl / ㎖) of the present invention.

Figure 5 is a diagram showing the inhibition of osteoclast proliferation by the mixed herbal medicine extract (Sukji sulfur water extract fraction (0.938 ~ 7.5μl / ㎖) and bone chain ethanol extract fraction (0.25 ~ 4.0μl / ㎖) of the present invention.

6 is a diagram showing the inhibition of osteoclast proliferation by the mixed herbal medicine extract (Sukjihwang butanol extract fraction (1.25 ~ 10.0μl / ㎖) and bone chain ethanol extract fraction (0.25 ~ 4.0μl / ㎖) of the present invention.

7 is a diagram showing the inhibition of osteoclast proliferation by the mixed herbal medicine extract (Sukjihwang ethanol extract fraction (0.625 ~ 5.0μl / ㎖) and bone chain ethanol extract fraction (0.25 ~ 4.0μl / ㎖) of the present invention.

The present invention relates to a mixed herbal extract of Sukji sulfur and bone chain beam and a composition for preventing and treating osteoporosis using the same as an active ingredient.

Osteoporosis is a disease caused by a weak balance between bone resorption and bone formation. It is caused by excessive bone resorption than bone formation. Osteoporosis is caused by a decrease in the amount of lime in bone tissue, resulting in thinning of the bone. The bone marrow cavity (骨髓 腔) is widened, and as the symptoms progress, the bones become weak, so even a small impact is likely to fracture. Bone tissue is a dynamic tissue that is formed by osteoblasts and constantly breaks down and absorbed by osteoclasts.

Osteoporosis is not a symptom itself but rather various fractures that are easily caused by bone weakness, especially femoral fractures or vertebral fractures, which limit long-term activity and lead to a healthy life, and consequently provide a cause for 15% of elderly deaths. It is known to do. 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 lower bone resorption levels than whites, resulting in higher bone mass. Usually bone mass is highest at 14-18 years of age 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 during menopause, in which a large amount of B-lymphocytes are produced as by interleukin-7 (IL-7), and the B cell precursor ( pre-B cells) accumulate and increase the amount of IL-6 thereby increasing the activity of osteoclasts.

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 an estimated $ 130 billion market in the field of bone disease treatment worldwide and is expected to grow further, so that many research institutes and pharmaceutical companies around the world are investing heavily in developing bone disease treatments. Currently, the development of a bone resorption inhibitor is actively underway.

In the past, the study was mainly focused on bone minerals, namely, calcium and phosphorus metabolic abnormalities, but no significant progress was made in determining the pathogenesis.

Materials currently used for the treatment of osteoporosis include bisphosphonate preparations (alendronate, ethidronate), hormone preparations (raloxifene), vitamin D preparations, calcitonin preparations, calcium preparations, and the like. However, bisphosphonate preparations are poorly absorbed, difficult to take and cause esophagitis. Hormonal preparations should be taken for a lifetime, and long-term administration may cause side effects such as breast cancer, uterine cancer, gallstones and thrombosis, and vitamin D preparations are expensive and effective. Unclear, calcitonin preparations are expensive and difficult to administer, and calcium preparations have fewer side effects but are limited to preventive effects rather than treatment.

Osteoporosis cannot be treated with short-term administration of the drug, but long-term administration of the drug is essential. Therefore, there is a need for a new substance that does not have such side effects and has good efficacy when the drug is administered for a long time.

On the other hand, Sukjihwang (Mature land sulfur; Rehmanniae Radix Preparata) is dried root of medicinal plants attached to Hyunsamaceae, and it is used as herbal medicine. Steamed and dried nine times to make it is called Gujihwang and the medicinal effect is the best. The taste is sweet but bitter and warm, and it supplements the blood and replenishes the blood (精: substance that is the basis for life and activity) and makes the waist and knees sore and painful symptoms, dysmenorrhea and dizziness. It has the effect of treating the back and blackening the head. Sukjihwang is the main medicine of Samul-tang (四 物 이며) and is used for symptoms such as heat resistance, dry throat, and thirst caused by the weakness of various chronic diseases. In folk medicine, taking pork with boiled broth is known to be effective for habitual constipation.

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

Accordingly, the present inventors, while conducting various physiological activity studies of Sukji sulfur and bone chain beam, the mixed herbal extract of Sookji sulfur and bone chain beam is not only toxic but also inhibits the growth of osteoclasts, thereby preventing and treating osteoporosis without side effects. The present invention has been completed by revealing the effectiveness thereof.

The present invention is to provide a mixed herbal medicine extract having osteoclast proliferation inhibitory activity mixed with sukji sulfur extract and bone chain extract.

In addition, the present invention is to provide a composition for preventing and treating osteoporosis using the mixed herbal medicine extract as an active ingredient.

The present invention provides a mixed herbal medicine extract having osteoclast proliferation inhibitory activity mixed with sukji sulfur extract and bone chain extract.

The present invention also provides a composition for the prevention and treatment of osteoporosis using the mixed herbal medicine extract as an active ingredient.

Compositions of the present invention include pharmaceutical and health food compositions useful for the prevention and treatment of osteoporosis.

Hereinafter, the present invention will be described in detail.

The present invention provides a herbal extract mixed with 0.15 ~ 15.0 parts by weight of Sukji sulfur extract and 0.05 ~ 4.0 parts by weight of bone chain beam extract.

The mixed herbal medicine included in the composition of the present invention is extracted and used with water, alcohol or an organic solvent, and the extraction method of the mixed herbal medicine is as follows.

Sukji sulfur and bone cladding were put into the extraction container, respectively, distilled water was added and extracted with hot water at 100 ℃ for 4 hours. After repeating the above procedure three times, the resulting solution is cooled to room temperature and filtered through a filter paper. Each of the extracts obtained was concentrated under reduced pressure at 40 ° C. or lower using a vacuum rotary evaporator to obtain a sukji sulfur extract and a bone chain extract, respectively. The concentrated Sukji sulfur extract is separated by bioassay-directed fractionation using butanol, ethyl acetate, ethanol. In addition, the concentrated bone scalp extract is separated by an activity tracking fraction using ethanol. The medicinal herb extract and golbolbo extract obtained in the above are mixed by 0.15 to 15.0 parts by weight and 0.05 to 4.0 parts by weight, respectively, to prepare a mixed herbal extract.

The present invention also provides a composition for the prevention and treatment of osteoporosis using the mixed herbal medicine extract as an active ingredient.

The mixed herbal medicine extract of the present invention significantly inhibits the production and activity of osteoclasts, compared to the case where Sukjihwang extract and Bolchibobo extract are treated alone, and can be effectively used for the prevention or treatment of osteoporosis.

The composition of the present invention may further contain at least one active ingredient exhibiting the same or similar function in addition to the mixed herbal medicine extract.

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 the like, which may be used in mixed herbal extracts, including at least one excipient such as starch, calcium carbonate, sucrose, lactose and gelatin, and the like. 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 varies depending on the patient's weight, age, sex, health condition, diet, time of administration, method of administration, excretion rate and severity of the disease, and the daily dosage is 200 to 400 mg / kg mixed herbal extract It is preferably 300 to 350 mg / kg, it may be administered 1 to 6 times a day.

As a result of conducting toxicity experiments by orally administering the mixed herbal extract of the present invention to rats, the 50% lethal dose (LD ₅₀ ) by the oral administration toxicity test is determined to be a safe substance of at least 1 g / kg or more.

The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the prevention and treatment of osteoporosis.

The composition of the present invention may be added to health food for the purpose of improving osteoporosis. When the mixed herbal medicine extract of the present invention is used as a food additive, the mixed herbal medicine extract may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient may be suitably determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, in the preparation of food or beverage, the mixed herbal extract of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less based on the raw material. However, in the case of long-term intake for health and hygiene or health control, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety. .

There is no particular limitation on the kind of food. Examples of the food to which the substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, drinks, Alcoholic beverages and vitamin complexes, and the like and include all of the health foods in the conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates, etc. as additional components, as in the general beverage. The above-mentioned natural carbohydrates are glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonic acid. Carbonating agents and the like used in beverages. In addition, the composition of the present invention may contain a flesh for preparing natural fruit juice, fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not critical but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, preferred examples and experimental examples are presented to help understand the present invention. However, the following Examples and Experimental Examples are provided only to more easily understand the present invention, and the contents of the present invention are not limited by the Examples.

Example  : Preparation of Mixed Herbal Extracts

1. Preparation of Sookjihwang Extract

1-1) Sukji sulfur water extraction fraction

100 g of Sookji sulfur was added to a 3 L extraction vessel, and 1 L of distilled water was added, followed by extraction of hot water at 100 ° C. for 4 hours. After repeating the above procedure three times, the resulting solution was cooled to room temperature and filtered through a filter paper. The extract was concentrated under reduced pressure at 40 ° C. or lower using a vacuum rotary evaporator to obtain a water extract fraction of sujiji sulfur (yield: 42%).

1-2) Sulfur Sulfur Butanol Extract

The concentrated Sukji sulfuric acid water extract obtained in 1-1) was put in a 1 L separatory funnel, 400 ml of n-butanol was added to remove internal pressure, and the upper layer was collected by fractionation of the solvent. This process was repeated three times to collect n-butanol fractions, and concentrated under reduced pressure at 40 ° C. or less using a vacuum rotary evaporator to remove n-butanol. After removing n-butanol, distilled water was added and dissolved (yield: 2.6%).

1-3) Sulfur Sulfur Ethyl Acetate Extraction Fraction

The concentrated Sukji sulfuric acid water extract obtained in 1-1) was placed in a 1 L separatory funnel, 400 ml of ethyl acetate was added to remove internal pressure, and the solvent was collected to collect an upper layer. This process was repeated three times to collect the ethyl acetate fractions, and concentrated under reduced pressure at 40 ° C. or lower using a vacuum rotary evaporator to remove ethyl acetate. Ethyl acetate was removed and distilled water was added for dissolution (yield: 0.4%).

1-4) Sulfur Sulfur Ethanol Extraction

400 ml of ethanol was added to the concentrated Sukji sulfuric acid extract fraction obtained in 1-1), adjusted to 80% ethanol, and then maintained at 4 ° C. in a refrigerator. The next day, except for the precipitate layer, the ethanol aqueous solution was filtered, and then concentrated under reduced pressure at 40 ℃ or less using a vacuum rotary evaporator to remove the ethanol. After ethanol was removed, distilled water was added to dissolve it (yield: 32.8%).

2. Preparation of Bone Clavicle Extract

2-1) Bone Clavicle Water Extraction Fraction

100 g of bone chain beam was placed in a 3 L extraction container, and 1 L of distilled water was added, followed by hot water extraction at 100 ° C. for 4 hours. After repeating the above procedure three times, the resulting solution was cooled to room temperature and filtered through a filter paper. The extract was concentrated under reduced pressure at 40 ° C or less using a vacuum rotary evaporator to obtain a bone chain extract extraction fraction (yield: 5.8%).

2-2) Bone Clambo Ethanol Extraction Fraction

400 ml of ethanol was added to the concentrated bone chain extract extract fraction obtained in 2-1), adjusted to 80% ethanol, and then maintained at 4 ° C. in a refrigerator. The next day, except for the precipitate layer, the ethanol aqueous solution was filtered, and then concentrated under reduced pressure at 40 ℃ or less using a vacuum rotary evaporator to remove the ethanol. After removing ethanol, distilled water was added and dissolved (yield: 1.8%).

3. Mixed Herbal Extract

3-1) Mixed Herbal Extract of Sukjihwang Water Extract Fraction and Bone Clavicle Extract Fraction

The mixed herbal medicine extract was prepared by mixing 0.15-4.9 parts by weight of the water extract fraction of Sukji sulfuric acid obtained in 1-1) and 0.05-4.0 parts by weight of the extract of bone chain beam obtained in 2-1) at an appropriate composition ratio.

3-2) Mixed medicinal herb extracts of Sookjihwang butanol extract fraction and bone chain supplement water extract fraction

0.5 ~ 14.7 parts by weight of the sulfuric acid butanol extract fraction obtained in 1-2) and 0.05 ~ 4.0 parts by weight of the extract of bone chain beam obtained in 2-1) were prepared in an appropriate composition ratio to prepare a mixed herbal extract.

3-3) Mixed Herbal Medicine Extract of Sulfur Sulfur Ethyl Acetate Extract Fraction and Bone Chain Beam Extract Fraction

Sucrose Sulfur Ethyl Acetate Extraction Fraction obtained in 1-3) and 0.25 ~ 6.67 parts by weight of Fractions extracted from 2-1) were extracted in an appropriate composition ratio to prepare a mixed herbal extract.

3-4) Mixed Herbal Extract of Sukjihwang Ethanol Extract Fraction and Bone Chain Extract Extract Fraction

Sulfur ethanol extract fraction 0.625 ~ 5.0 parts by weight obtained in 1-4) and 0.25 ~ 2.0 parts by weight of the bone chain extract extract fraction obtained in 2-1) was mixed at an appropriate composition ratio to prepare a mixed herbal extract.

3-5) Mixed Herbal Extract of Sukjihwang Water Extract Fraction and Bone Chain Beam Ethanol Extract Fraction

The mixed herbal medicine extract was prepared by mixing 0.938 to 7.5 parts by weight of the water extract fraction of Sukji sulfuric acid obtained in 1-1) and 0.25 to 4.0 parts by weight of the ethanol extract fraction of bone chain obtained in 2-2).

3-6) Mixed Herbal Extracts of Sookjihwang Butanol Extract Fraction and Bone Chain Beam Ethanol Extract Fraction

Sukji sulfur butanol extract fraction obtained in 1-2) and 1.25 ~ 10.0 parts by weight of the ethanol extract fraction Bolchibori obtained in 2-2) was mixed in an appropriate composition ratio to prepare a mixed herbal extract.

3-7) Mixed Herbal Medicine Extract of Sulfur Sulfur Ethanol Extract Fraction and Bone Chain Beam Ethanol Extract Fraction

Sulfur ethanol extract fraction 0.625 ~ 5.0 parts by weight obtained in 1-4) and 0.25 ~ 4.0 parts by weight of the ethanol extract fraction Bolchibobo obtained in 2-2) was prepared by mixing the appropriate herbal extract.

The mixed herbal extracts were diluted in the culture and used for the following experiments.

Experimental Example 1  : Proliferation inhibitory effect of osteoclasts (osteoclast) by the mixed herbal medicine extract of the present invention

In order to examine how the mixed herbal medicine extract of the present invention affects the growth and activity of osteoclasts, osteoclast progenitor cells were cultured on calcium-phosphate-coated plates (OAAS, OCT Inc.) TRAP (Tartrate-resistant acid phosphatase; hereinafter abbreviated as "TRAP") activity was analyzed.

1. Pure separation of osteoclast progenitor cells and differentiation into mature osteoclasts

In order to separate the mouse bone marrow cells, 7 to 9 weeks old male mice were sacrificed with cervical torsion, followed by sterile extraction of the femur and tibia, removal of soft tissues, and the cutting of both ends of the iliac bone. 1 ml of enzyme solution containing 0.1% collagenase (Gibco), 0.05% trypsin and 0.5 mM EDTA (Gibco) was injected into the bone marrow cavity to remove the bone marrow, stirred for 30 minutes, and the bone marrow cells were collected to contain 10% FBS. After incubation in α-MEM (α-minimum essential medium) for 24 hours, unattached cells were collected. Unattached cells, which become progenitor cells of osteoclasts, were cultured by aliquoting 2 × 10 ⁵ cells per well. Mixed herbal medicine of the above embodiment in α-MEM containing 20ng / ml macrophage-colony stimulating factor (M-CSF, Peprotech, USA) and 30ng / ml RANKL (Peprotech, USA) during 8 days of culture The extract was treated and incubated. After incubation, the cells were fixed and subjected to TRAP staining to examine the production of osteoclasts.

2. Measurement of Multinucleated Cell Formation as TRAP (+)

The measurement of osteoclast formation was observed by the number of multinucleated cells showing TRAP (+) after incubating osteoclast progenitor cells on a plate coated with calcium-phosphate.

Specifically, after the cell culture, the adherent cells were washed with PBS, and then fixed with citrate-acetate-formaldehyde for 5 minutes, naphthol AS-BI phosphate, fast garnet ( fast Garnet) TRAP staining by incubating for 1 hour in 37 ℃ acetate buffer (pH 5.0) containing GBC solution and 7mM tartrate buffer (pH 5). TRAP (+) multinucleated cells with three or more nuclei were considered as osteoclasts.

1) The results of the osteoclast proliferation inhibition rate (%) of the water extract fraction of Sookji sulfur and the bone extract water extract fraction are shown in Table 1 and FIG. 1.

Osteoclast proliferation inhibition rate (%) Concentration (μl / ml) Fractured Water Extract 0 0.05 0.15 0.44 1.33 4.0 Sukjiwang Water Extract 0 0 10.0 22.0 38.0 60.2 76.0 0.15 4.0 7.6 17.5 28.8 69.8 84.6 0.5 10.0 28.0 22.3 40.9 66.7 90.3 1.6 22.5 46.3 50.0 62.1 82.2 93.6 4.9 78.3 94.0 93.0 95.7 98.3 100.0

As shown in Table 1 and Figure 1, when the Sukji sulfur water fraction was treated alone, the inhibition rate of the control group was 0 and the inhibition rate of the number of TRAP-positive multinucleated cells compared to the control group was 0.15, 0.5, 1.6, and 4.9 μl, respectively. The production and activity of osteoclasts were inhibited at 4.0, 10.0, 22.5, 78.3% at the concentration of / ml. In addition, when the bone chain extract fraction was treated alone, the formation and activity of osteoclasts were inhibited at 10.0, 22.0, 38.0, 60.2, and 76.0% at concentrations of 0.05, 0.15, 0.44, 1.33, and 4.0 μl / ml, respectively.

The expected osteoclast proliferation inhibition rate of the mixed herbal medicine extract indicates the inhibition rate in the rest except the inhibition rate of the herbal medicine extract alone. Therefore, as a result of Table 1, the osteoclast inhibition ability of the mixed medicinal herb extract of Sukjihwang water extract fraction and Bonechibo water extract fraction was higher than that of Sukjihwang water extract fraction or Bonechibo water extract fraction. And it can be seen that the activity is significantly inhibited. Particularly, the treatment of the combination of 0.15 and 0.5 μl / ml of the Sulfur Sulfur water extract fraction and 1.33 and 4.0 μl / ml of the Bulchulbo water extract fraction and 0.05 μl / mL of the Sulphur Water extract fraction 1.6 and 4.9 μl / ml When combined concentrations of ml or more were treated, the production and activity of osteoclasts was significantly suppressed compared with the treatment alone.

2) The results of inhibition rate (%) of osteoclast proliferation of the butanol butanol extract fraction and the bone chain extract extract is shown in Table 2 and FIG.

Osteoclast proliferation inhibition rate (%) Concentration (μl / ml) Fractured Water Extract 0 0.05 0.15 0.44 1.33 4.0 Sulfur Sulfur Butanol Extract 0 0 10.0 22.0 38.0 60.2 76.0 0.5 15.0 10.0 10.6 35.1 74.1 89.0 1.6 30.0 30.0 48.5 66.0 78.9 93.0 4.9 50.0 50.1 62.5 72.7 82.3 95.6 14.7 97.7 98.7 100.0 100.0 99.3 100.0

As shown in Table 2 and FIG. 2, the control ratio of the control group was 0 when the sulfuric acid butanol extract fraction was treated alone, and the inhibition ratio of the number of TRAP positive multinucleated cells compared to the control group was 0.5, 1.6, 4.9, and 14.7 μl, respectively. 15.0, 30.0, 50.0, 97.7% at the concentration of / ml inhibited the production and activity of osteoclasts. In addition, when the bone chain extract fraction was treated alone, the formation and activity of osteoclasts were inhibited at 10.0, 22.0, 38.0, 60.2, and 76.0% at concentrations of 0.05, 0.15, 0.44, 1.33, and 4.0 μl / ml, respectively. Particularly, the case of treatment of a combination of 0.5 μl / ml of sulfuric acid butanol extract fraction and 1.33 and 4.0 μl / ml of bone chain extract fraction and 0.15 μl / ml or more of the sulfuric acid butanol extract fraction 1.6 and 4.9 μl / ml and bone fragment extract fraction Treatment with a combination of concentrations significantly inhibited the production and activity of osteoclasts compared with treatment with each concentration alone.

3) The results of inhibition rate (%) of osteoclast growth of the ethyl acetate extract fraction and sucrose bone extract extract fractions are shown in Table 3 and FIG.

Osteoclast proliferation inhibition rate (%) Concentration (μl / ml) Fractured Water Extract 0 0.05 0.15 0.44 1.33 4.0 Sulfur Sulfur Ethyl Acetate Extract 0 0 10.0 22.0 38.0 60.2 76.0 0.25 10.0 10.0 9.6 26.7 56.0 81.6 0.74 22.5 15.0 38.5 59.8 78.3 88.3 2.22 35.0 24.0 49.7 56.5 78.0 92.7 6.67 60.0 64.6 72.8 78.3 84.7 96.6

As shown in Table 3 and FIG. 3, the inhibition rate of the control group was 0 and the inhibition rate of the number of TRAP positive multinucleated cells compared to the control group was 0.25, 0.74, 2.22, and 6.67, respectively. The production and activity of the osteoclasts were inhibited at 10.0, 22.5, 35.0, and 60.0% at the concentration of [mu] l / ml. In addition, when the bone chain extract fraction was treated alone, the formation and activity of osteoclasts were inhibited at 10.0, 22.0, 38.0, 60.2, and 76.0% at concentrations of 0.05, 0.15, 0.44, 1.33, and 4.0 μl / ml, respectively. Particularly, in the case of the treatment of a combination of 0.25 μl / ml of the sulfuric acid ethyl acetate extract fraction and 4.0 μl / ml of the bone chain extract fraction, the sulfuric acid ethyl acetate extract fraction of 0.74 and 2.22 μl / ml and the bone fragment extract fraction of 0.15 μl / ml In the case of treatment with a combination of concentrations, and treatment with a combination of concentrations of 6.67 μl / ml of the sulfuric acid ethyl acetate extract fraction and 0.05 μl / ml of the bone chain extract fraction, the production of osteoclasts was compared with the treatment alone. Activity was significantly inhibited.

4) The results of the osteoclast proliferation inhibition rate (%) of the ethanol extract fraction of Sookji Hwang and the bone chain extract extract are shown in Table 4 and FIG. 4.

Osteoclast proliferation inhibition rate (%) Concentration (μl / ml) Fractured Water Extract 0 0.25 0.5 1.0 2.0 Sulfur Sulfur Ethanol Extract 0 0 6.05 11.50 12.07 19.80 0.625 43.86 36.99 30.97 46.44 90.26 1.25 36.70 35.27 45.29 74.51 95.70 2.5 82.81 93.70 98.00 98.57 100.00 5.0 100.00 100.00 100.00 100.00 100.0

As shown in Table 4 and FIG. 4, when the ethanol extract fraction of Sukji was treated alone, the inhibition rate of the control group was 0 and the inhibition rate of the number of TRAP-positive multinucleated cells compared to the control group was 0.625, 1.25, 2.5, 5.0 μl, respectively. 43.86, 36.70, 82.81, 100.0% at the concentration of / ml inhibited the production and activity of osteoclasts. In addition, when the bone chain extract fraction was treated alone, the formation and activity of osteoclasts were inhibited at 6.05, 11.50, 12.07, and 19.8% at concentrations of 0.25, 0.5, 1.0, and 2.0 μl / ml, respectively. In particular, the treatment of the combination of 0.625 μl / ml of the sulfuric acid ethanol extract fraction and 2.0 μl / ml of the bone chain extract extract fraction and 1.25 μl / ml of the sulfuric acid ethanol extract fraction and 1.0 and 2.0 μl / ml or higher bone extract extract fraction When treated in combination, the production and activity of osteoclasts was significantly suppressed compared to the case of treatment alone.

5) The results of the osteoclast growth inhibition rate (%) of Sukji sulfur water extract fraction and bone chain ethanol extract fraction are shown in Table 5 and FIG.

Osteoclast proliferation inhibition rate (%) Concentration (μl / ml) Bone Clavicle Ethanol Extraction Fraction 0 0.25 0.5 1.0 2.0 4.0 Sukjiwang Water Extract 0 0 0.0 11.78 16.37 15.79 34.98 0.938 59.33 53.60 49.59 61.91 47.87 49.30 1.875 67.06 72.50 73.65 69.93 67.35 82.24 3.75 96.85 93.41 98.57 96.28 99.01 99.14 7.5 100.00 100.00 100.00 100.00 100.00 100.0

As shown in Table 5 and FIG. 5, the inhibition rate of the control group was set to 0, and the inhibition rate of the number of TRAP-positive multinucleated cells compared to the control group was 0.938, 1.875, 3.75, and 7.5, respectively. The production and activity of the osteoclasts were inhibited at 59.33, 67.06, 96.85, and 100.0% at the concentration of [mu] l / ml. In addition, when the bone chain ethanol extract fractions were treated alone, the formation and activity of osteoclasts were inhibited at 0.0, 11.78, 16.37, 15.79, and 34.98% at concentrations of 0.25, 0.5, 1.0, 2.0, and 4.0 μl / ml, respectively. Particularly, the treatment of Sukji sulfur water extract fraction 1.875 ul / ml and the bone chain ethanol extract fraction 0.25 and 0.5 ul / ml were combined with the concentration of 3.75 ul / ml Sukji sulfur water extract fraction and 0.5 ul / ml bone bran extract ethanol extract fraction. When treated in combination, the production and activity of osteoclasts was significantly suppressed compared to the case of treatment alone.

6) The results of inhibition rate (%) of osteoclast proliferation of the sulfuric acid butanol extract fraction and the bone chain ethanol extract fraction are shown in Table 6 and FIG. 6.

Osteoclast proliferation inhibition rate (%) Concentration (μl / ml) Bone Clavicle Ethanol Extraction Fraction 0 0.25 0.5 1.0 2.0 4.0 Sulfur Sulfur Butanol Extract 0 0 0.0 11.78 16.37 15.79 34.98 1.25 26.68 9.78 28.97 12.07 33.55 54.17 2.5 56.18 43.00 47.30 49.59 40.14 78.52 5.0 89.69 84.25 88.54 89.69 93.99 98.28 10.0 100.00 100.00 100.00 100.00 100.00 100.0

As shown in Table 6 and Figure 6, when treated with Sukji sulfur butanol extract alone, the inhibition rate of the control group was set to 0 and the inhibition rate of the number of TRAP positive multinucleated cells compared to the control group was 1.25, 2.5, 5.0, and 10.0 μl, respectively. The production and activity of osteoclasts were inhibited at 26.68, 56.18, 89.69, and 100.0% at the concentration of / ml. In addition, when the bone chain ethanol extract fractions were treated alone, the formation and activity of osteoclasts were inhibited at 0.0, 11.78, 16.37, 15.79, and 34.98% at concentrations of 0.25, 0.5, 1.0, 2.0, and 4.0 μl / ml, respectively. In particular, when treated with a combination of 5.0 μl / ml and 2.0 and 4.0 μl / ml bone ethanol extract fraction of Sookji-sulfur butanol fraction, osteoclast production and activity were significantly suppressed compared to the treatment alone.

7) The results of the osteoclast proliferation inhibition rate (%) of the ethanol extract fraction of Sookji sulfur and the ethanol extract fraction of the bone chains are shown in Table 7 and FIG. 7.

Osteoclast proliferation inhibition rate (%) Concentration (μl / ml) Bone Clambo Ethanol Extract 0 0.25 0.5 1.0 2.0 4.0 Sulfur Sulfur Ethanol Extract 0 0 0.0 11.78 16.37 15.79 34.98 0.625 43.86 26.68 36.99 33.55 32.41 57.61 1.25 36.70 44.15 43.58 62.77 67.06 87.68 2.5 82.81 93.99 97.42 99.43 98.28 100.00 5.0 100.00 100.00 100.00 100.00 100.00 100.00

As shown in Table 7 and FIG. 7, the inhibition rate of the control group was 0 and the inhibition rate of the number of TRAP-positive multinucleated cells compared to the control group was 0.625, 1.25, 2.5, 5.0 μl when the ethanol extract fraction of Sukji was treated alone. 43.86, 36.70, 82.81, 100.00% at the concentration of / ml inhibited the production and activity of osteoclasts. In addition, when the bone crushed ethanol extract fraction was treated alone, the formation and activity of osteoclasts were inhibited at 0.0, 11.78, 16.37, 15.79, 34.98% at concentrations of 0.25, 0.5, 1.0, 2.0 and 4.0 μl / ml, respectively. . Particularly, when the sulfuric acid ethanol extract fraction 1.25 µl / ml and the bone chain ethanol extract fraction 2.0 and 4.0 µl / ml were treated in combination, and the sulfuric acid ethanol extract fraction 1.25 and 2.5 µl / ml and the bone chain ethanol extract fraction 0.25 µl / ml When the combinations of the above concentrations were treated, the production and activity of the osteoclasts was significantly suppressed as compared with the treatment alone.

Therefore, it can be seen that the mixed herbal medicine extract of the present invention significantly inhibits the production and activity of osteoclasts as compared to the case where Sukjihwang extract and Bolchibobo extract are treated alone.

Experimental Example 2  : Acute Toxicity of Oral Administration in Rats

In order to determine the acute toxicity of the mixed herbal medicine extract of the present invention, an acute toxicity test was carried out by the following method.

Acute toxicity test was conducted using 6-week-old specific pathogen-free (SPF) SD rats. The mixed herbal extracts of Sukjihwang water extract fraction and Bolchibobo water extract fraction prepared in Example 3-1) were suspended in 0.5% methyl cellulose solution in 2 animals per group, and used once at a dose of 1 g / kg / ml. Oral administration. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were examined. Hematological and hematological examinations were performed. Necropsy was performed to visually observe the abdominal organ 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 any toxic change even in rats up to 1 g / kg, and the minimum lethal dose (LD ₅₀ ) of oral administration was determined to be a safe substance of more than 1 g / kg of extract.

Examples of preparations for the compositions of the present invention are illustrated below.

The mixed herbal medicine extract of Sukjihwang water extract fraction and Bolchibobo water extract fraction prepared in Example 3-1) was used in the preparation example.

Formulation Example 1  : Preparation of Pharmaceutical Formulations

1. Preparation of powder

Mixed Herbal Extract 2g

1g lactose

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

2. Preparation of Tablets

Mixed Herbal Extract 100mg

Corn Starch 100mg

Lactose 100mg

2 mg magnesium stearate

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

3. Preparation of Capsule

Mixed Herbal Extract 100mg

Corn Starch 100mg

Lactose 100mg

2 mg magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

Formulation Example 2  : Manufacture of food

Food products containing the mixed herbal medicine extract of the present invention were prepared as follows.

1. Preparation of Cooking Seasonings

20 to 95% by weight of the mixed herbal medicine extract of the present invention was prepared for cooking spices for health promotion.

2. Preparation of Tomato Ketchup and Sauce

0.2 ~ 1.0% by weight of the mixed herbal medicine extract of the present invention was added to tomato ketchup or sauce to prepare tomato ketchup or sauce for health promotion.

3. Manufacturing of Flour Foods

0.5 ~ 5.0% by weight of the mixed herbal medicine extract of the present invention was added to the flour, and using the mixture to prepare bread, cakes, cookies, crackers and noodles to prepare a health promoting food.

4. Preparation of soups and gravy

The mixed herbal medicine extract of the present invention was added to the soup and broth to prepare a health-promoting meat products, soup of noodles and broth.

5. Preparation of Ground Beef

10% by weight of the mixed herbal medicine extract of the present invention was added to ground beef to prepare ground beef for health promotion.

6. Manufacture of Dairy Products

5 ~ 10% by weight of the mixed herbal medicine extract of the present invention was added to the milk, using the milk to prepare a variety of dairy products such as butter and ice cream.

7. Manufacture of wire

Brown rice, barley, glutinous rice, and yulmu were alphad by a known method, and then dried and roasted to prepare a powder having a particle size of 60 mesh using a grinder.

Black beans, black sesame seeds, and perilla were also steamed and dried by a known method, and then ground to a powder having a particle size of 60 mesh.

The mixed herbal medicine extract of the present invention was decompressed and concentrated in a vacuum concentrator, and the dried product obtained by drying with a spray and a hot air dryer was pulverized with a particle size of 60 mesh to obtain a dry powder.

It was prepared by combining the dry powder of the grains, seeds and mixed herbal medicines prepared in the following ratio.

Cereals (30% by weight brown rice, 15% by weight radish, 20% by weight barley),

Seeds (7% by weight perilla, 8% by weight black beans, 7% by weight black sesame),

Dry powder (3% by weight) of the mixed herbal medicine extract,

Ganoderma lucidum (0.5% by weight),

Foxglove (0.5 wt%)

Formulation Example 3  : Preparation of Beverages

1. Preparation of carbonated drinks

5-10% of sugar, 0.05-0.3% citric acid, 0.005-0.02% caramel, 0.1-1% of vitamin C are mixed and 79-94% purified water is mixed to make syrup, and the syrup is 85-98 Sterilizing for 20 ~ 180 seconds at ℃ and mixed with a cooling water in a ratio of 1: 4 and then injected with 0.5 ~ 0.82% carbon dioxide gas to prepare a carbonated beverage containing the mixed herbal medicine extract of the present invention.

2. Manufacture of health drinks

Instant sterilization by homogeneously mixing subsidiary ingredients such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5%), water (75%) and mixed herbal extracts , Packed in a small packaging container such as plastic bottles to prepare a healthy beverage.

3. Preparation of Vegetable Juice

5 g of the mixed herbal medicine extract of the present invention was added to 1,000 ml of tomato or carrot juice to prepare vegetable juice for health promotion.

4. Preparation of Fruit Juice

1 g of the mixed herbal medicine extract of the present invention was added to 1,000 ml of apple or grape juice to prepare fruit juice for health promotion.

The mixed herbal medicine extract of the present invention effectively inhibits the formation and activity of osteoclasts, and thus can be widely used as medicines and health foods useful for the prevention and treatment of osteoporosis.

Claims (9)

       A pharmaceutical composition for osteoporosis prevention and treatment, comprising a medicinal herb extract, which has been mixed in a weight ratio of 0.15 to 15.0: 0.05 to 4.0, as the active ingredient. delete The composition according to claim 1, having an osteoclast proliferation inhibitory activity. delete delete         The composition of claim 1, wherein the sperm sulphide extract is a water extract.         The composition according to claim 1, wherein the succinate sulfur extract is a solvent fraction of any one selected from the group consisting of ethanol, butanol and ethyl acetate of the succinate sulfur water extract.         The composition of claim 1, wherein the Golbobo extract is a water extract of Golbobo.        The composition of claim 1, wherein the Golbobo extract is an ethanol solvent fraction of the Golbobo water extract.

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