KR102411098B1 - Composition for preventing, ameliorating or treating osteoporosis containing natural product material extract comprising Achyranthes japonica as effective component - Google Patents

Abstract

The present invention relates to a composition for the prevention, improvement or treatment of osteoporosis, comprising a mixed extract mixed with hyssopus extract, duchung alcohol extract and Angelica asiatica as an active ingredient, wherein the mixed extract has a remarkable TRAP activity inhibitory effect, The composition of the present invention comprising the mixed extract may be usefully used as a therapeutic agent for osteoporosis or a health functional food composition for anti-osteoporosis.

Description

A composition for preventing, improving or treating osteoporosis, comprising an extract of a natural material containing hyssop as an active ingredient, the composition for preventing, improving or treating osteoporosis containing natural product material extract comprising Achyranthes japonica as effective component

The present invention relates to a composition for preventing, improving, or treating osteoporosis, comprising as an active ingredient a mixed extract obtained by mixing a hyssop extract, an eucalyptus alcohol extract, and an Angelica asiatica extract.

With the change of the osteoporosis treatment paradigm, the main fields of development of osteoporosis drugs are research and development in the fields of osteoporosis mechanisms, osteoporosis control, and osteoporosis diseases. The field of osteoporosis mechanism studies the differentiation and activity of osteoclasts and osteoblasts, and the field of osteoporosis control involves various hormones, cytokines, antibodies, drug delivery methods involved in bone metabolism, and inhibition of osteoclasts and osteoblasts by bone regeneration cells. Research on the enhancement of bone density through cell promotion, and research on targets and novel therapeutic substances for osteoporosis-related diseases in the field of osteoporosis disease. In the treatment of osteoporosis, there are bisphosphonates, estrogen, calcitonin, etc. as bone resorption inhibitors, and bisphosphonate preparations, which account for about 80% of the total treatment market, are used as representative drugs in the treatment of osteoporosis. However, some side effects such as jaw bone necrosis and atypical femur fractures occur, and even with treatment, it can only be used for about 5 years.

Parathyroid hormone has been approved for use as an agent that promotes bone formation. It is also used limitedly in patients with severe osteoporosis of the spine and is expensive, and the maximum treatment period is limited to 2 years due to concerns about the occurrence of bone cancer. As a treatment for osteoporosis in postmenopausal women, estrogen supplementation, a hormone replacement therapy, was attempted and reported to be effective in maintaining bone mass. However, in the elderly aged 60 years or older, the effect of inhibiting bone loss is low, and long-term treatment is required for at least 5 years, and complications such as uterine bleeding, endometrial cancer, and breast cancer occur.

Therefore, there is a need for an osteoporosis improving agent using natural materials that have fewer side effects and are easily accessible, with fewer side effects, as the osteoporosis treatment paradigm itself creates new bone beyond preventing bone loss.

It is known that certain components such as saponins from natural medicinal plants and ginseng derived from natural products containing polyphenols and flavonoids with antioxidant activity inhibit osteoclast differentiation and function to function as a bone resorption inhibitor. An object of the present invention is to provide a functional food composition for preventing and treating osteoporosis by increasing bone density by minimizing bone resorption by inhibiting osteoclast activity using safe natural materials of effective medicinal plants with such antioxidant components.

Korea Patent No. 2320679 discloses a composition for preventing or treating osteoporosis comprising a complex extract of phalanx phalanx and hyssopus as an active ingredient, and Korean Patent Publication No. 2017-0015446 discloses osteoporosis comprising a hyssop extract and ginseng extract as active ingredients. Although the composition for prevention and treatment is disclosed, it is different from the composition for prevention, improvement or treatment of osteoporosis comprising the extract of a natural material containing hyssop as an active ingredient of the present invention.

The present invention has been devised in response to the above needs, and an object of the present invention is to provide a composition for preventing, improving or treating osteoporosis, which contains a mixed extract mixed with hyssopus extract, duchung alcohol extract, and Angelica asiatica alcohol extract as an active ingredient. The present invention was completed by confirming that the composition inhibits osteoclast differentiation of osteoclasts has a significant synergistic effect as compared to the hyssop extract, the Duchung alcohol extract and the Angelica asiatica extract alone.

In order to solve the above problems, the present invention provides a health functional food composition for the prevention or improvement of osteoporosis comprising a mixed extract mixed with hyssopus extract, duchung alcohol extract, and angelica asiatica extract as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of osteoporosis, comprising a mixed extract mixed with hyssopus extract, duchung alcohol extract, and Angelica asiatica extract as an active ingredient.

The present invention relates to a composition for the prevention, improvement or treatment of osteoporosis, comprising, as an active ingredient, a mixed extract obtained by mixing a hyssopus extract, a duchung alcohol extract, and an Angelica asiatica extract. In comparison, the TRAP activity inhibitory effect of the mixed extract is the most remarkable, and it is determined that the mixed extract can be usefully used as a therapeutic agent for osteoporosis or as a health functional food for preventing or improving osteoporosis.

1 is a graph comparing cytotoxicity to MC3T3-E1 cells after induction of inflammation of wooseul (Ach), duchung (Euc), angelica (Ang), and longanyuk (Dim) water extracts.
Figure 2 is a graph comparing the cytotoxicity to MC3T3-E1 cells after inflammation induction of wooseul (Ach), duchung (Euc), angelica (Ang), and longanyuk (Dim) alcohol extract.
3 is a graph comparing the osteoclast differentiation activity of water extracts of wooseul (Ach), duchung (Euc), angelica (Ang), and longanyuk (Dim).
Figure 4 is a graph comparing the osteoclast differentiation activity of wooseul (Ach), duchung (Euc), angelica (Ang), and longanyuk (Dim) alcohol extract.
Figure 5 shows the cytotoxicity and osteoclast differentiation activity of a mixture (MIX-A) and a mixture (MIX-B) in which the extracts of Urseul, Angelica, and Duchung are mixed in a 1:1:1 ratio. is a graph comparing
MIX-A concentration: 1000 = hyssop 100 µg/ml + Angelica humus 100 µg/ml + Duchong 100 µg/ml, 500 = hyssopus 50 µg/ml + Angelica 50 µg/ml + Duchong 50 µg/ml, 250 = Ursula 25 µg /ml + Angelica 25 µg/ml + Duchung 25 µg/ml, 100 = Urseul 10 µg/ml + Angelica 10 µg/ml + Duchung 10 µg/ml
MIX-B concentration: 1000 = hyssop 100 μg/ml + angelica 200 μg/ml + duchung 200 μg/ml, 500 = hyssop 50 μg/ml + angelica 100 μg/ml + duchung 100 μg/ml, 250 = hyssop 25 μg /ml + Angelica 50 μg/ml + Duchung 50 μg/ml, 100 = Wooseul 12.5 μg/ml + Angelica 25 μg/ml + Duchung 25 μg/ml
6 is a TRAP solution stained by treatment of a mixture (MIX-A) mixed with an extract of Urseul, Angelica keis, and Duchung alcohol extracts in a 1:1:1 ratio (MIX-A) and a mixture (MIX-B) mixed in a 1:2:2 ratio This is a photograph of the treated cells observed under an optical microscope.

In order to achieve the object of the present invention, the present invention provides a health functional food composition for the prevention or improvement of osteoporosis, comprising as an active ingredient a mixed extract mixed with hyssopus alcohol extract, duchung alcohol extract, and Angelica asiatica extract.

In one embodiment of the present invention, the mixed extract is preferably a hyssop extract at a concentration of 40 to 60 μg/ml, an alcohol extract from Duchung at a concentration of 40 to 60 μg/ml, and an alcohol extract from Angelica asiatica at a concentration of 40 to 60 μg/ml. may be a mixed extract mixed in a volume ratio of 0.8 to 1.2:0.8 to 2.2:0.8 to 2.2, and more preferably, a 50 μg/ml concentration of hyssop extract, a 50 μg/ml concentration of Duchung alcohol extract, and 50 μg/ It may be a mixed extract obtained by mixing the alcohol extract of Angelica biloba at a concentration of ㎖ in a volume ratio of 1:1:1 or 1:2:2.

In addition, in one embodiment of the present invention, the alcohol extract preferably contains 60 to 80% (v/v) alcohol 8 to 12 times (v/w), and then at 60 to 80° C. for 6 to 10 hours. It may be extracted, and more preferably 70% (v/v) alcohol is added 10 times (v/w) and then extracted at 70° C. for 8 hours, but is not limited thereto.

In addition, in one embodiment of the present invention, the mixed extract is characterized in that it inhibits the formation of osteoclasts.

The health functional food composition of the present invention is preferably prepared in the form of powder, granule, pill, tablet, capsule, candy, syrup or beverage, but is not limited thereto.

When the health functional food composition of the present invention is used as a food additive, the active ingredient may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient may be suitably determined according to the purpose of its use (prevention, health or therapeutic treatment). In general, in the production of food or beverage, the composition of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts 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 less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount greater than the above range.

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

When the composition of the present invention is used as a health drink, it may contain various flavoring agents or natural carbohydrates as an additional component like a conventional drink. The above-mentioned natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclotenstrin, and sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetener, natural sweeteners such as taumatine and stevia extract, synthetic sweeteners such as saccharin and aspartamic acid, and the like can be used. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 g 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 agents, pH regulators, stabilizers, preservatives, glycerin, alcohol , a carbonation agent used in carbonated beverages, and the like. In addition, the composition of the present invention may contain pulp for the production of natural fruit juice, fruit juice beverage, and vegetable beverage. These components may be used independently or in combination. The proportion of these additives is not very important, but the composition of the present invention is generally selected in the range of 001 to 01 parts by weight per 100 parts by weight.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of osteoporosis, comprising a mixed extract mixed with hyssopus extract, duchung alcohol extract, and Angelica asiatica extract as an active ingredient.

In addition to the active ingredient, a pharmaceutically acceptable carrier, excipient or diluent may be further included, and various oral or parenteral formulations may be used. In the case of formulation, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include capsules, powders, granules, tablets, pills, etc., and such solid preparations include one or more compounds and at least one excipient, for example, starch, calcium carbonate, sucrose or lactose ( lactose), gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral administration include suspensions, emulsions, syrups, aerosols, and the like, and various excipients such as wetting agents, sweetening agents, fragrances, preservatives, etc. may be included 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, freeze-dried preparations, and suppositories. As the non-aqueous solvent and the suspending solvent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycero gelatin, etc. may be used. For parenteral administration, it is preferable to select external or intraperitoneal, rectal, intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebrovascular injection.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the level of the effective amount is determined by the type, severity, and drug activity of the patient. , can be determined according to factors including sensitivity to drug, administration time, administration route and excretion rate, duration of treatment, concurrent drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by a person skilled in the art.

The dosage of the composition of the present invention varies depending on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and severity of disease. 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.

Hereinafter, the present invention will be described in more detail using examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited thereto.

Preparation Example 1. Plant Alcoholic Extract

70% alcohol was added to 10 times (v/w) of the raw material to hyssop (Ach), duchung (Euc), and angelfish (Ang), extracted under reflux for 8 hours at 70°C, concentrated under reduced pressure, and freeze-dried hyssop alcohol extract; Duchung alcohol extract and Angelica asiatica alcohol extract were prepared, respectively.

The prepared hyssop alcohol extract, Duchung alcohol extract, and Angelica asiatica extract were mixed in a 1:1:1 volume ratio (50 Brix).

Preparation Example 2. Stick-type beverage pouch

(1) 8 times (v/w) of purified water compared to ground red ginseng was added and extracted at 95° C. for 8 hours to prepare a red ginseng extract. After filtering the prepared red ginseng extract, it was concentrated at 60° C. to prepare a 65-70 brix red ginseng concentrate.

(2) based on the total weight of the stick-type beverage, 30% by weight of the plant alcohol extract of Preparation Example 1, 5% by weight of the red ginseng concentrate prepared in step (1) and 0.2% by weight of the complex golden extract, 20% by weight of fructooligosaccharide, After mixing 12% by weight of cyclodextrin syrup, 3% by weight of N-acetylglucosamine and 27.8% by weight of purified water, 10 g of each was filled in a stick.

Preparation Example 3. Pill

(1) 8 times (v/w) of purified water compared to ground red ginseng was added and extracted at 95° C. for 8 hours to prepare a red ginseng extract. After filtering the prepared red ginseng extract, it was concentrated at 60° C. to prepare a 65-70 brix red ginseng concentrate.

(2) Based on the total weight of the dough, 30% by weight of the plant alcohol extract of Preparation Example 1, 3% by weight of the red ginseng concentrate prepared in step (1), 25% by weight of crystalline cellulose, 20% by weight of rice flour, 15% by weight of fructooligosaccharide Weight%, N- acetylglucosamine 3% by weight, silicon dioxide (SiO ₂ ) 2% by weight and whey calcium 2% by weight were mixed to prepare a round dough.

(3) The dough prepared in step (2) was molded into a ring with a ventilation machine, dried, and packaged to contain 3 g of the prepared ring per package.

1. Material extraction

1) Alcoholic extract

70% alcohol was added 10 times (v/w) of the raw material to oxul (Ach), duchung (Euc), angelfish (Ang), and longan meat (Dim), extracted under reflux at 70°C for 8 hours, concentrated under reduced pressure, and frozen Experiments were conducted with dried hyssop extract, duchung alcohol extract, angelica asiatica alcohol extract, and longan meat alcohol extract.

2) water extract

10 times (v/w) of the raw water was added to each of hyssop (Ach), Duchung (Euc), Angelica (Ang), and Longan Meat (Dim), extracted under reflux at 88°C for 8 hours, concentrated under reduced pressure, and freeze-dried hyssop. Experiments were conducted with water extract, duchung water extract, angelica water extract, and longan meat water extract.

2. Experimental method

1) Molecular biological measurement for evaluation of osteogenic efficacy

A) Cell culture

Human-derived osteoblast line MG63 cells were purchased from American Type Culture Collection (ATCC; Rockville, MD, USA) for evaluation of cell compatibility of osteoblasts, 10% FBS and 1% penicillin-streptomycin (100 U/ml, 100 μg/ml) ) in DMEM-high glucose medium containing 5% CO ₂ , and subcultured in an incubator at 37° C. at intervals of 3 days.

B) Osteoblast survival rate (MTT assay)

MC3T3-E1 cells were seeded in a 96-well plate at a concentration of 1×10 ⁴ cells/well and stabilized in DMEM containing 10% FBS and 1% penicillin-streptomycin for 24 hours. After adding osteoblastic differentiation reagents (OS, 1M 2-glycerophosphate, 50 μg/ml ascorbic acid, and 100nM dexamethasone) to the same medium, 0, 50, 100, 200, and 500 μg/ml of each sample After treatment with a concentration of 5% CO ₂ , incubated for 24 hours in an incubator at 37 ℃. After incubation, 50 μl of MTT solution (2 mg) was added and reacted in an incubator at 5% CO ₂ and 37° C. for 2 hours, then the medium was removed, and 150 μl of DMSO was added and reacted for 20 minutes at room temperature. After dissolving the formazan crystal, absorbance was measured at 540 nm using a microplate reader (BIO-RAD 450, California, USA).

C) Alkaline phosphatase activity measurement (ALP assay)

MC3T3-E1 cells were aliquoted to a density of 2×10 ⁴ ml/well in a 6-well plate and stabilized in DMEM containing 10% FBS and 1% penicillin-streptomycin for 24 hours. Osteoblastic differentiation reagents (OS, 1M 2-glycerophosphate, 50 μg/ml ascorbic acid, and 100nM dexamethasone) were added to the same medium, and each sample was treated by concentration, 5% CO ² , 37°C Incubated in an incubator for 7 days, the culture medium including the sample was replaced 3 times a week, then the cells were harvested, dissolved with 0.1% triton X-100 at 4°C for 1 hour, and centrifuged at 13,000 rpm and 4°C for 10 minutes. Collect the supernatant, mix each sample supernatant and substrate solution [15mM p-NPP (p-nitrophenyl phosphate)] in equal amounts and react in an incubator at 37°C for 30 minutes. After the reaction was terminated by the addition of 0.9N NaOH (50 μg/ml), the absorbance was measured at 405 nm. Activity was measured from a standard curve prepared using 1 mM p-NP (p-nitrophenol), and the protein concentration of the sample was quantified through the BCA protein assay method, and then the value was corrected and expressed as nmol/30min/mg of protein.

3) Molecular biological measurement to evaluate osteoclast differentiation effectiveness

A) Osteoclast culture and cell viability evaluation (MTT assay)

RAW264.7 cells were seeded in a 96-well plate at a concentration of 1×10 ⁴ cells/well, and RANKL (50 ng/ml) was added to α-MEM supplemented with 10% FBS and 1% penicillin-streptomycin to induce osteoclast differentiation. ) was added. The concentration of the sample was divided into 0, 50, 100, and 500 μg/ml, and the group not treated with each extract was set as a control group, and after 3 days of culture, MTT solution (2 mg) was added to 5% CO ₂ , 37 After reacting in an incubator at ℃ for 2 hours, 150 μl of DMSO was treated, left at room temperature for 20 minutes, and absorbance was measured at 540 nm using a microplate reader.

B) Observation of osteoclasts: Observation of mature osteoclasts with TRAP (tartate resistant acid phosphate) staining

Cells 3 days old with differentiation agent and sample treatment were washed with PBS, fixed with 10% formalin for 5 minutes, washed again with DW, and stained with Sigma TRAP (kit no. 387; Sigma-Aldrich, St. Louis. MO, USA) Treated with fresh TRAP solution for 30 minutes according to the method, the stained cells were observed with an optical microscope, and the image was imaged with a digital imaging camera.

TRAP activity analysis: TRAP assay (in order to measure the activity of the osteoclast-specific marker TRAP enzyme, TRAP solution assay is performed by Tintut et al.)

Differentiation agent and sample treatment After washing the cells with water for 3 days, 80 μl of cold lysis buffer (90 mM citrate, pH 4.8, 0.1% Triton X-100 containing 80 mM sodium tartrate) was dispensed and treated for 10 minutes. Substrate solution (20 mM p -nitrophenyl phosphate) was added by 80 μl, 5% CO ₂ , reacted for 20 minutes in an incubator at 37° C., and the reaction was stopped with 40 μl of 0.5N NaOH, at 405 nm using an ELISA reader. Absorbance was confirmed.

4) Statistical processing

All experimental values in this study were repeated three or more times, and the results were expressed as mean ± standard deviation. For statistics, two-way ANOVA (Analysis of Variation) test was performed, then the multi-range test of Bonferroni post tests and the two-tailed Student's t test were performed. to verify the significance at the p <0.05 level.

5) Analysis of hyssop, duchung, and angelica index substances

The analysis conditions of the hyssop index component (20-Hydroxyecdysone) are shown in Table 1 below.

Wooseul Index Component Analysis Conditions equipment HPLC Agilent 1260 series detector UV (245 nm) column Prontosil 120-5-C18-ace-EPS
(4.6 mm × 250 mm, 5.0 μm) column temperature 30℃ mobile phase 0.08% acetonitrile formate mixture (17:3) flow rate 1.0 ml/min infusion dose 10 μl

Table 2 below shows the conditions for analysis of the cephalothorax component (Pinoresinol diglucoside).

Conditions for analysis of cephalothorax indices equipment HPLC Agilent 1260 series detector UV (230 nm) column Prontosil 120-5-C18-ace-EPS
(4.6 mm × 250 mm, 5.0 μm) column temperature 35℃ mobile phase Mobile phase A-0.1% formic acid in water
Mobile phase B-Acetonitrile hours (minutes) Mobile phase A (%) Mobile phase B (%) 0 95 5 5 95 5 25 30 70 30 30 70 flow rate 1.0 ml/min infusion dose 10 μl

Angelica index component (Decursin) analysis conditions are shown in Table 3 below.

Angelica Decursin Analysis Conditions equipment HPLC Agilent 1260 series detector UV (330 nm) column Prontosil 120-5-C18-ace-EPS
(4.6 mm × 250 mm, 5.0 μm) column temperature 25℃ mobile phase Mobile phase A-Acetonitrile
Mobile phase BD.W. hours (minutes) Mobile phase A (%) Mobile phase B (%) 0 20 80 3 20 80 8 30 70 18 30 70 19 50 50 40 50 50 41 90 10 50 90 10 flow rate 1.0 ml/min infusion dose 10 μl

Example 1. osteoclast toxicity

The results of the cytotoxicity test for MC3T3-E1 cells are shown in FIGS. 1 and 2 . In the cytotoxicity according to the concentration of water extracts of hyssop (Ach), duchung (Euc), angelfish (Ang), and longanyuk (Dim), angelic guinea and longanyuk did not show cytotoxicity at any concentration, and hyssop at a concentration of 200 μg/ml or higher, duchung showed cytotoxicity at a concentration of 500 μg/ml ( FIG. 1 ). In the case of the alcohol extract, longan meat did not show cytotoxicity at any concentration, and hyssopus, Angelica and Duchung showed cytotoxicity at a concentration of 500 μg/ml ( FIG. 2 ).

Example 2. Induction of osteoclasts from raw materials (RANKL 50 ng/ml treat)

As a result of the inhibitory activity of osteoclast differentiation activity, the ethanol extract showed inhibition of osteoclast differentiation rather than the water extract except for longan meat. At the same concentration (100 μg/ml) of the hyssopus, Angelica, and Duchung extracts at the cell safety level, the Duchong alcohol extract showed the best inhibitory ability ( FIGS. 3 and 4 ). Compared to the concentration of each raw material alone, it showed very excellent activity in all of the mixed raw materials (FIG. 5). As a result of TRAP staining, round-shaped osteoclasts (ROC), ie, TRAP-positive multinucleated osteoclasts, were clearly observed in the control group, and osteoclast formation was significantly inhibited in all of the mixed materials ( FIG. 6 ).

When the above results are taken together, at a concentration that does not show cytotoxicity, a significant TRAP production inhibitory ability was obtained at the same concentration of Mixed A (50 μg/ml) compared to extracts of hyssopus, Angelica, and Duchung alone (50 μg/ml). indicated. In addition, it was found that there was no significant difference in inhibitory ability between Mix B and Mix A, which had higher concentrations of Angelica and Duchung during the preparation of the mixed extract.

Inhibitory effect on osteoclast differentiation by concentration: Inhibition of TRAP production type TRAP activity (%) Hyssop extract (50 μg/mL of hyssop) 60.9 Angelica lily extract (Anggui 50 ㎍/mL) 85.5 Duchung alcohol extract (Ducong 50 μg/mL) 60.6 Mixed A (MIX-A)* 29.0 Mix B (MIX-B) 28.8

*MIX-A: Hyssop 50 μg/ml + Angelica 50 μg/ml + Duchung 50 μg/ml

MIX-B: Wooseul 50 μg/ml + Angelica 100 μg/ml + Duchung 100 μg/ml

Example 3. Indicator material analysis result

As a result of analyzing the indicator substances of the hyssopus, Angelica, and Duchung alcohol extracts, the hyssop extract contained 0.899 mg/g of Hydroxyecdysone, and the Angelica spirit extract contained 17.105 mg/g of decursin, and the alcohol extract of Duchung. , pinoresinol diglucoside was detected at a content of 5.177 mg/g.

Indicator material analysis result type indicator substance Content (mg/g) Hyssop Spirit Extract Hydroxyecdysone 0.899 Angelica Root Extract Decursin 17.105 Duchung Alcoholic Extract pinoresinol diglucoside 5.177

Claims (7)

A mixed extract of 40-60 ㎍/㎖ concentration of hyssop extract, 40-60 ㎍/㎖ concentration of Duchung alcohol extract, and 40-60 ㎍/㎖ concentration of Angelica asiatica alcohol extract in a 1:1:1 volume ratio is effective A health functional food composition for the prevention or improvement of osteoporosis comprising as a component. delete The method according to claim 1, wherein each of the alcohol extracts is an extract extracted at 60-80°C for 6-10 hours after adding 60-80% (v/v) alcohol 8-12 times (v/w). A health functional food composition for the prevention or improvement of osteoporosis. The health functional food composition for preventing or improving osteoporosis according to claim 1, wherein the mixed extract inhibits the formation of osteoclasts. According to claim 1, wherein the composition is a health functional food composition for the prevention or improvement of osteoporosis, characterized in that it is prepared in any one formulation selected from powder, granules, pills, tablets, capsules, candy, syrup and beverages. A mixed extract of 40-60 ㎍/㎖ concentration of hyssop extract, 40-60 ㎍/㎖ concentration of Duchung alcohol extract, and 40-60 ㎍/㎖ concentration of Angelica asiatica alcohol extract in a 1:1:1 volume ratio is effective A pharmaceutical composition for the prevention or treatment of osteoporosis comprising as a component. The pharmaceutical composition for preventing or treating osteoporosis according to claim 6, further comprising a pharmaceutically acceptable carrier, excipient or diluent in addition to the mixed extract.

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