KR20070018489A - A pharmaceutical composition comprising the extract of herb mixture for treating or preventing osteoporosis disease - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention and treatment of osteoporosis disease, including a mixed herbal extract, more specifically, seomoktae ( Phynchosia nulubillis ; rat eye ) and golden extract as an active ingredient, in addition to the extract anchovy , Pharmaceutical composition for the prevention and treatment of osteoporosis comprising one or more selected from the group consisting of black sesame, seaweed, kelp, safflower seed, perilla, royal jelly, osteoporosis, degenerative bone disease and prevention of bone diseases such as rheumatoid arthritis And useful for treatment.

Osteoporosis, Golden Extract, Seomoktae (Rice Eye Bean), Pharmaceutical Composition, Health Functional Food

Description

Pharmaceutical composition comprising the extract of herb mixture for treating or preventing osteoporosis disease

1 is a diagram illustrating an experiment schedule for carrying out an animal experiment of the present invention.

Figure 2a to Figure 2b shows the weight change after feeding the F formula of the present invention, Figure 2c is a result of measuring the organ weight of each group after induction of osteoporosis due to ovarian extraction and low calcium diet.

Figure 3 shows the results of measuring the tibia and femur weight of each group according to the present invention.

Figure 4 is a result of measuring the concentration of metabolites in the serum according to the present invention, Figure 4a is ALP activity, Figure 4b is the concentration of inorganic phosphorus, Figure 4c shows the concentration of calcium.

Figure 5 shows the histological density change of the trabecular (trabecular) according to the present invention.

Figure 6 is a photograph showing a histological density change of the trabecular bone (trabecular) according to the invention, Figure 6a is a bone density after osteoporosis induced, Figure 6b is after administration of the formula F of the present invention for 4 weeks, Figure 6c Is a photograph after 8 weeks of administration.

The present invention relates to a pharmaceutical composition for the prevention and treatment of osteoporosis comprising a mixed herbal extract, more specifically, Seoktaetae ( Phynchosia nulubillis ; rat eye ) and golden extract as an active ingredient, in addition to the extract anchovy, black sesame, It relates to a pharmaceutical composition for the prevention and treatment of osteoporosis comprising one or more selected from the group consisting of seaweed, kelp, safflower seed, perilla, royal jelly.

Osteoporosis is a condition in which bone density is markedly reduced compared to normal people of the same age and sex, and the cause of the invention is known to be involved in endocrine, nutritional and genetic factors (Gauley et al., Am. J. Epidemiol , 124 , p752, 1986). In particular, the decrease in bone density with menopause is more severe in women, and the incidence of osteoporosis is significantly higher than in men, and research on this is being actively conducted.

As a treatment for postmenopausal osteoporosis, estrogen supplementation, a hormone replacement therapy, has been attempted and is reported to be effective in maintaining bone mass. However, this method of treatment is less effective than the early menopause in elderly people over 60 years of age, requires at least five years of long-term treatment, risk of irregular uterine bleeding, breast cancer, endometrial cancer, and high blood pressure It is pointed out that there is.

Meanwhile, studies on the treatment of osteoporosis using calcium and vitamin D, calcitonin, bisphosphate, androgen, and flavonoids have been conducted, but none of the drugs can fully restore the reduced bone mass. No, the treatment is also limited. Therefore, osteoporosis is emphasized the importance of prevention rather than treatment after onset.

Recently, researches on alternative therapies using natural products such as foods as active ingredients have been actively conducted to compensate for the dangers caused by the administration of estrogen. As a representative alternative therapy for the prevention and treatment of postmenopausal osteoporosis, oral administration of phyroestrogen or ingestion of a food containing a large amount thereof has been attempted. Phytoestrogens are contained in alfafa, peas, oatmeal, red beans, rice, and soybeans. In particular, isoflavone-based phytoestrogens are contained in legumes. Isoflavones have been identified 12 types such as aglucon genistein, daidzein, glycidin, and glycosides in which sugars are bound, and among these, dizein and genistein Has a structure similar to that of estrogen, and it is found to bind to the estrogen receptor in the cell and simultaneously exhibit not only an estrogen effect but also an antiestrogenic effect. As a result, it has been in the spotlight as an estrogen replacement material for the prevention of osteoporosis due to the advantage that it does not cause various side effects by the administration of estrogen.

As such, claims to supplement isoflavones in the West have been realized, and studies on the relationship between osteoporosis and quantitative concentrations of isoflavones in the blood and urine of osteoporosis patients are also underway.

As described above, soybeans are widely used in the West for the prevention and treatment of osteoporosis, while rat or soybean (seomoktae), anchovy, sesame, seaweed, safflower and the like have been used in oriental medicine and folk medicine. However, scientific studies of drugs associated with osteoporosis have only reported that soy extract induces increased bone growth or increased osteoblast proliferation in ovariectomized rats, but that contains large amounts of isoflavone-based phytoestrogens. The research on is poor. In addition, there is no study on the effect of diet containing the main functional ingredients that have an effective function in osteoporosis.

Meanwhile, golden ( Scutellaria baicalensis ) is a dried root of the pericarp herbaceous perennial herb ( Scutellaria baicalensis GEORGI) belonging to the Lamiaceae, is less toxic and the main component is separated from 30 species of flavonoid compounds. These pharmacological actions of gold include antipyretic, diuretic, antiviral, antibacterial, sedative, blood pressure lowering, blood sugar elevation, edema (利 膽 作用), intestinal (腸管) exercise, anaphylaxis Anaphylaxis, anti-inflammatory, anti-allergic, anti-oxidant and cell-immune-promoting functions are used in herbal medicines, which are used in over 70 kinds of golden baths as commercial prescription drugs.

Of the three flavonoids (baicalin, baicalein and wogonin), the roots of gold, baicalein was most relieved of inflammatory symptoms in inflammatory animal models compared to other flavonoids. sulfasalazone) has been reported to be similar to the efficacy of 50 mg / kg.

On the other hand, in the case of baicalin, the inhibitory effect on carrageenan-induced rat paw eduma and antitumor effect using bladder cancer cells showed that the inhibition of the proliferation of cancer cells was higher than that of baicalein and wogonin. It was reported. The chemical structure of baicalin is that the hydroxy group of baicalein is replaced by the glucuronyloxy group, which is thought to cause different pharmacological action between the two components.

wogonin selectively inhibits the expression of COX-2 in RAW 264.7 cells, a macrophage cell line in mice, inhibits the action of inducible macrophage-type nitric oxide synthase (iNOS) and reduces PGE ₂ production, respectively. The effect of inhibiting production has been reported. In addition, the expression of COX-2 was selectively inhibited when applied 5 times at 50 to 200 ㎍ / site / treatment concentration in the skin of mice induced with inflammation by TAP (12-O-tetradecanoylphoral-13-acetate). At the same time, it has been reported to be effective in preventing and treating inflammation by inhibiting PGE ₂ production.

As described above, the study of a single component of gold is partially made, but the study of gold itself has not been disclosed or taught.

Therefore, the present inventors have made diligent efforts to secure a substance capable of treating osteoporosis from traditional herbal medicines that are relatively safe. As a result, the golden extract of the present invention confirms the therapeutic effect of inflammation caused by osteoporosis in addition to the herbal pharmacological theory. To complete.

After all, the main object of the present invention is to provide a mixed herbal extract having a therapeutic and prophylactic effect of osteoporosis disease.

In addition, another object of the present invention to provide a pharmaceutical composition for the prevention and treatment of osteoporosis disease comprising the mixed herbal extract as an active ingredient.

In order to achieve the above object, the present invention provides a mixed herbal extract comprising a mouse eye bean (seomoktae) containing a large amount of isoflavone-based phytoestrogens and golden extract showing the effects of antioxidant and anti-inflammatory action.

The mixed herbal medicine is characterized in that it comprises one or more selected from the group consisting of anchovy, black sesame, seaweed, kelp, safflower seed, perilla, royal jelly in addition to the rat bean and golden extract.

The mixed herbal extract of the present invention includes water and C ₁ to C ₄ lower alcohols such as methanol, ethanol, butanol and the like, and a mixture thereof, preferably an extract available in methanol.

Hereinafter, the present invention will be described in detail.

The mixed herbal extract according to the present invention can be obtained as follows.

Polar solvents of C ₁ to C ₄ lower alcohols, such as methanol, ethanol, butanol, and the like, about 1 to 30 times, preferably 3 to 20 times the weight of the herbal drug sample of the present invention or about 1: The supernatant was recovered by vacuum filtration using an extraction method such as hot water extraction, cold immersion extraction, reflux cooling extraction, or ultrasonic extraction with a mixed solvent having a mixing ratio of 0.1 to 1:10, and then the above procedure was repeated several times. The supernatant can be collected and concentrated under reduced pressure to obtain the herbal extract.

The present invention provides a pharmaceutical composition for the prevention and treatment of osteoporosis disease containing the herbal extract obtained by the above-mentioned method as an active ingredient. The herbal medicine of the present invention has been used as a herbal medicine for a long time, the extracts of the present invention extracted therefrom also have no problems such as toxicity and side effects.

The pharmaceutical composition for preventing and treating osteoporosis disease according to the present invention comprises 0.1 to 50% by weight of the extract based on the total weight of the composition.

The pharmaceutical composition comprising the extract of the present invention may further comprise suitable tints, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

The pharmaceutical dosage forms of the extracts of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable collection.

Pharmaceutical compositions comprising extracts according to the invention, in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterile injectable solutions, respectively, according to conventional methods. Can be formulated and used. Carriers, excipients and diluents that may be included in the composition comprising the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, aceitol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid preparations may contain at least one excipient such as starch, calcium carbonate and sucrose in the extract. Or lactose, gelatin, or the like is mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, liquid 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. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

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

The present invention provides a health functional food comprising the extract and a food-acceptable food supplement additive exhibiting a prophylactic effect of osteoporosis disease.

Examples of the food to which the mixed herbal medicine extract of the present invention can be added include various foods, beverages, gums, teas, vitamin complexes, and health functional foods.

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

The health functional beverage composition of the present invention is not particularly limited to other ingredients in addition to containing the extract as an essential ingredient in the indicated ratio, and may contain additional ingredients such as various flavoring agents or natural carbohydrates as in general beverages. . Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a flavoring agent other than the above-mentioned, a natural flavoring agent, taumartin, a stevia extract, for example, rebaudioside A, glycyrrhizin, etc .; And synthetic flavoring agents such as saccharin, aspartame and the like. The proportion of such natural carbohydrates is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the extract of the present invention has various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavors, coloring and neutralizing agents (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective properties Colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the extracts of the present invention may contain flesh for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. At this time, the ratio of the additive is not very important, but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the extract of the present invention.

Hereinafter, the present invention will be described in more detail based on the following Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, but the content of the present invention is not limited thereto.

Example 1 Preparation of Rat Eye Extract

1 kg of rat eye bean (Seomok Tae) was washed with distilled water and powdered with a crusher, and 5 L of ethanol was added thereto, extracted for 1 week at room temperature, and the supernatant was recovered by vacuum filtration. The process was repeated three times to collect the supernatant, and then concentrated under reduced pressure and then freeze-dried for 72 hours to obtain rat eye extract.

Example 2. Preparation of Golden Extract

Gold was purchased and used in Yeosan, Jeollanam-do, Yeocheon. 2 g of water was added to 100 g of golden roots, followed by repeated hot water extraction with a shaker at 120 ° C. for 2 hours, concentrated under reduced pressure, and freeze-dried the concentrate to obtain a golden extract.

Example 3. Preparation of K Extract

10 g of anchovy, black sesame, seaweed, kelp, safflower seed, perilla were added to distilled water and extracted with hot water for 6 hours, then cooled to room temperature and allowed to stand for one day to remove precipitates.

Example 4 Preparation of F Diet

The rat eye extract, golden extract and K extract prepared in Examples 1 to 3 were mixed at the same ratio (weight ratio), and with respect to 1 kg of the mixture, 20 mg of CPPII and 50 mg of condroitin sulfate as an additive. Formula F was prepared by combining 50 mg of glucosamine, 50 mg of collagen, 50 mg of shark cartilage, 50 mg of vitamin C, 70 mg of algae calcium, 10 mg of L-Lysin HCl, and 10 mg of isoflavone.

Experimental Example 1. Experiment Preparation

1-1. Experimental animals

Sprague-Dawely female rats were purchased from Samtaco Bio Korea (Osan, Korea) and adapted to the laboratory environment for approximately three weeks with sufficient supply of solid compressed feed and water for rats. After the weight, the ovary extraction was carried out when the body weight was 200-300 g, and classified into control group, sham group (comparative group), OVX group and F-diet group (experimental group), respectively.

Ovarian extraction was performed by mixing ketara (ketamine hydrochloride, Yuhan Corporation) and lumpoon injection solution (Bay Korea) at a rate of 2 ml / kg BW and 0.4 ml / kg BW, respectively, and injecting into the abdominal cavity for general anesthesia, and then removing the dorsal hair. After excision, skin and muscles were excised to expose the ovary, ligation of the fallopian tubes with thread, the ovaries were excised, the muscles were sutured with the remaining thread, and the skin was sutured with an animal stapler. The ovary was also extracted on the other side in the same way. In this case, surgery was performed in the same manner, but the ovary was performed without the ovary and a sham operator was used as a comparison group.

1-2. Preparation of Experimental Diet and Administration of Final Diet

After extraction of the ovary, as shown in Table 1 below, the experimental diet was prepared, and the control group was fed to the control group without the ovary and drug administration, the control group (sham group) and ovary OVX group and F-diet (experimental group), which had been extracted, were given a low calcium diet to induce osteoporosis. The diet was carried out for 4 weeks after ovarian extraction.

TABLE 1

ingredient Control Low Calcium Diet Casein 200 200 DL-Methionine 3 3 Sucrose 424.9 431.1 Corn starch 150 150 Corn oil 100 100 cellulose 50 50 Vitamin mixtures 10 10 Mineral mixtures 35 - Mineral mixtures (except Ca and P) - 35 Calcium Carbonate (CaCO ₃ ) 5 5 Choline chloride 2 2 Calcium Phosphate (CaHPO ₄ · 2H ₂ O) 12.02 1.72 Potassium Phosphate (KH ₂ PO ₄ ) 8.08 16.22 Sum 1000 1004.04

The final diet was administered orally to the F diet of the present invention prepared in Example 4 of 2 mg per 100 g body weight once every 8 weeks (F- diet group), sham group and OVX group 1 per 100 g body weight daily ML of distilled water was orally administered.

Experimental Example 2. Weight Measurement

Body weight was measured using an electronic scale once a week just before ovarian extraction and after ovarian extraction.

Experimental Example 3. Calculation of food efficiency ratio (FER)

After feeding the experimental diet, feed intake was measured twice a week, and the dietary efficiency was calculated by dividing the weight gain during the breeding period by the diet intake during the same period.

Experimental Example 4. Measurement of organ, tibia and femur weight and bone density

After completion of the breeding period, the animals were anesthetized with ethyl ether, and then opened, and blood was collected through the liver portal vein. Various organs, tibia, and femur were collected.

The collected blood was left at room temperature for 30 minutes, centrifuged at 3000 rpm for 15 minutes to obtain serum, and frozen and stored at -70 ° C. Immediately after blood collection, liver, kidney and spleen were separated, washed with physiological saline, and filtered After removing the water with a weighing was stored frozen.

In addition, the tibia and femur were collected and weighed to measure bone density.

Figure 2a shows the weight change after 4 weeks of feeding the F formula of the present invention, Figure 2b shows the weight change after 8 weeks. In addition, Figure 2c is a result of measuring the organ weight of each group after ovarian extraction and osteoporosis-induced by the supply of low calcium diet and after feeding the F formula of the present invention.

As shown in Figures 2a and 2b, the sham group, OVX group and F-die group all showed weight gain, with sham group 28.67 ± 22.62 g, OVX group 76.67 ± 28.29 g and F- diet group 106.33 ± 42.77 g In particular, significant weight gain was observed in the other groups compared to the sham group.

As a result of long-term weight measurement after osteoporosis induction, liver weight was in the order of OVX group> sham group> control group, and height was OVX group> control group> sham group, and sham group> control group> OVX group in spleen.

However, after 4 weeks of feeding the F diet of the present invention, the change in the weight of the organs was simple F-diet group> sham group> OVX group, and in case of kidney, sham group> OVX group> F-diet group and spleen weight of F-diet Group> sham group> OVX group. On the other hand, after feeding the F formula of the present invention for 8 weeks, the weight of the organ was measured. The liver weight was F-diet group> sham group> OVX group, and the weight of kidney was F-diet group> sham group> OVX group, spleen In the case of F-diet> OVX group> sham group (see Figure 2c).

The results of measuring the tibia and femur weight of each group are summarized in FIG. 3. As can be seen in Figure 3, the weight of the tibia of each group after osteoporosis induction was in the order of the control> sham group> OVX group, and the femur was in the order of sham group> OVX group> control. On the other hand, after administering the F diet of the present invention for 4 weeks, the tibial weight was in the order of sham group> OVX group> F-diet group, and the femur was in the order of F-diet group> OVX group> sham group. However, after 8 weeks, the tibia and femur weights were confirmed in the order of F-diet> OVX> sham group.

That is, the weight of the femur of the group fed the F diet of the present invention for 4 weeks was significantly increased compared to the other groups, especially after 8 weeks it was confirmed that the tibia and femur showed a significant increase.

Experimental Example 5. Measurement of Metabolites in Serum

Reagents for measuring metabolites in serum were measured using an automatic analyzer of Bio clinical system corporation, and absorbance was measured using BECKMEN DU530.

5-1. Alkaline phosphatase (ALP) measurement

For the determination of alkaline phosphatase in serum, 2 ml of substrate buffer was warmed for 5 minutes in a 37 ° C constant temperature water bath using a Kind-king method, and 0.05 ml of the sample was mixed well, followed by warming for 15 minutes in a constant temperature water bath for 10 minutes at room temperature. Absorption was measured at 500 nm as a control within 60 minutes after standing.

5-2. Inorganic Measurement

Measurement of inorganic phosphorus in serum was performed using the molybdenum pigment method using a phosphorus kit (Bio clinical system corporation).

0.02 ml of the sample was well mixed with 8 ml of the color reagent, and the mixture was left at room temperature for 30 minutes, and then absorbance was measured using the reagent blind as a control at a wavelength of 650 nm.

5-3. Calcium measurement

Calcium in blood is measured by OCPC method.Measurement reagent is mixed with 5 ml of buffer with 0.5 ml of color reagent using a calcium kit (bio clinical system corporation). After standing at room temperature for 5 minutes, the absorbance was measured by blinding within 90 minutes as a control.

Each measurement result was put together in FIGS. 4A-4C.

In terms of ALP activity, OVX group showed higher ALP activity than sham group after osteoporosis induction. However, four weeks after the F diet, the F-diet group decreased compared to the OVX group, and even after eight weeks, the F-diet group showed the order of sham group> OVX group> F-diet group (see FIG. 4A).

The concentration of inorganic phosphorus in blood was highest in OVX group after osteoporosis induction, followed by sham group and control group, but 4 weeks after F diet of the present invention, F-diet> sham group> OVX group. After 8 weeks, OVX group> F-diet group> sham group appeared in order, and there was no significant difference between groups (see FIG. 4B).

On the other hand, the concentration of calcium in the blood was sham group> OVX group> control group after osteoporosis induction, F- diet group> sham group> OVX group, 8 weeks after sham group> F diet administration of the present invention> OVX group> F-diet group appeared in order, but also there was no significant difference between groups (see Figure 4c).

Experimental Example 6. Measurement of bone mineral density (BMD)

Tibia was collected from rats of each group, fixed with Bouin's solution (or 10% formalin buffer solution), dehydrated with 5% nitric acid solution for 5-7 days, washed with running water for 12 hours, and then paraffin embedded through normal tissue specimen preparation. After embedding, a 5 μm thick tissue specimen was prepared using a microtop, followed by Gomori staining.

Tibia was placed in a tube in which 70% alcohol of XCT research SA (STRATEC, Germany) was locked and fixed to the hold to check the overall shape of the tibia through SV scan, and the desired position was selected and analyzed by CT at the selected position. .

FIG. 5 shows the density of trabecular bones at 2 mm below the end of the bone site of the osteoporosis-induced osteoporosis of rats, and it was confirmed that osteoporosis was clearly induced in the OVX group compared to the control and sham groups. .

Four weeks after the F meal of the present invention, there was no significant difference between the total density and the trabecular density. However, after 8 weeks there was a tendency to be significantly improved compared to the control.

The above results can be confirmed also in Figures 6a to 6c showing the histological density change of the bone graft according to the F-form administration of the present invention, illustrating that the F-form of the present invention is effective in the prevention and treatment of osteoporosis disease.

Experimental Example 7. Measurement of proliferation of osteoblasts and osteoblasts

DMG with MG-63 human osteoblast attached to a polystyrene cell culture dish and added 1% antibacterial-antifungal solution containing penicillin and streptomycin (Gibco, USA) and 10% FBS (Gibco, USA) (Gibco, USA) was incubated with 5% CO ₂ while maintaining a humidity of 95%, temperature 37 ℃.

The proliferation of osteoblasts can be measured in a short period of time with the mass search of MTS [3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxy-methoxyohenyl) -2- (4-sulfophenyl) -2H-tetra- zolim)] was measured using a colorimetric assay method (Cell Titer 96 Aqueous One Solution Cell Proliferation Assay: Promega). After dispensing at a concentration of 0.5 ㅧ 104 cell / well in a 96 well plate, and after 24 hours, rats and soybean methanol extract, E2, diazein, and genistein were incubated for 5 days.

On the fifth day, add MTS (30 μl / well) reagent, incubate at 37 ° C. for 2 hours 30 minutes, and measure the absorbance at 490 nm using an ELISA reader to determine the amount of MTS that is decomposed into formazan. Decided.

Each treatment group secreted 12 wells, repeated three times, and the effect of cell proliferation on each drug was expressed as a percentage of the control group treated with DMSO only after taking the average of three replicates.

As can be seen in Table 2 showing the results, it was confirmed that the F formula of the present invention is effective for osteoblast proliferation.

TABLE 2

Therapy Proliferation (%) Control 100 F meal (10 mg / ml) 142.5

Experimental Example 8. Statistical Processing

Statistical method-mean ± SD was used. Statistical processing between the two groups was performed by Student's t-test, and the p-value less than 0.05 was considered as a significant difference.

One example of the formulation of the pharmaceutical composition comprising the extract of the present invention, but the present invention is not intended to limit it, but is intended to explain in detail.

Formulation Example 1 Preparation of Powder

Mixed Herbal Extract Powder 20mg

Lactose 100 mg

Talc 10 mg

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

Formulation Example 2 Preparation of Tablet

Mixed Herbal Medicine Extract Powder 10mg

100 mg corn starch

Lactose 100 mg

2 mg magnesium stearate

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

Formulation Example 3 Preparation of Capsule

Mixed Herbal Medicine Extract Powder 10mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 2mg

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

Formulation Example 4 Preparation of Injection

Mixed Herbal Medicine Extract Powder 10mg

Mannitol 180 mg

Sterile distilled water for injection 2794 mg

_{Na 2 HPO 4 · 12H 2 O} 26 ㎎

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

Formulation Example 5 Preparation of Liquid

Mixed Herbal Medicine Extract Powder 10mg

10 g of isomerized sugar

5 g of mannitol

Purified water

According to the conventional method of preparing a liquid solution, each component is added and dissolved in purified water, lemon flavor is added to the appropriate amount, the above components are mixed, and then purified water is added to adjust the total amount to 100 ml, and then sterilized by filling in a brown bottle. To prepare.

Formulation Example 6 Preparation of Healthy Drink

Mixed Herbal Medicine Extract Powder 10mg

15 g of vitamin C

100 g of vitamin E (powder)

Iron lactate 19.75 g

3.5 g of zinc oxide

Nicotinamide 3.5 g

0.2 g of vitamin A

0.25 g of vitamin B1

0.3 g of vitamin B2

Water

After mixing the above components in accordance with a conventional method for preparing a healthy beverage, and then stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and refrigerated and stored in the present invention It is used to prepare a healthy beverage composition according to.

Although the composition ratio is mixed with a component suitable for a preferred beverage in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and usage.

As described above, the mixed herbal extract of the present invention suppresses weight gain, exhibits a significant increase in osteoporosis of the tibia and the femur, and proliferative effects of osteoblasts. It can be used as a pharmaceutical composition for the prevention and treatment of diseases.

In addition, the mixed herbal medicine of the present invention can be usefully used as a health functional food by using natural substances without toxicity and side effects.

Claims (9)

Mixed herbal extracts containing Phynchosia nulubillis and golden extract as active ingredients. The method of claim 1, In addition to the extract, mixed herbal medicine extract comprising one or more selected from the group consisting of anchovy, black sesame, seaweed, kelp, safflower seed, perilla, royal jelly. A pharmaceutical composition for the prevention and treatment of osteoporosis diseases containing Phynchosia nulubillis and golden extract as active ingredients. The method of claim 3, wherein In addition to the extract, pharmaceutical composition for the prevention and treatment of osteoporosis disease comprising one or more selected from the group consisting of anchovy, black sesame, seaweed, kelp, safflower seed, perilla, royal jelly. The method of claim 3, wherein The extract is a pharmaceutical composition that is an extract available in water, lower alcohols of C1 to C4 or a mixed solvent thereof. The method of claim 3, wherein The osteoporosis disease is osteoporosis, degenerative bone disease or rheumatoid arthritis pharmaceutical composition. A dietary supplement comprising Phynchosia nulubillis ( capsicum bean), a golden extract, and a food-acceptable food supplement, which have an effect of preventing and improving osteoporosis diseases. The method of claim 7, wherein In addition to the extract, health functional food comprising one or more selected from the group consisting of anchovy, black sesame, seaweed, kelp, safflower seed, perilla, royal jelly. The method of claim 7, wherein Health functional food which is health drink.

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