A COMPOSITION COMPRISING COMBINED EXTRACTS OF SCHISANDRA FRUCTUS, EUCOMMIAE CORTEX AND LYCII FRUCTUS FOR PREVENTING OR TREATING METABOLIC BONE DISEASES

Abstract

The present invention provides a pharmaceutical composition or a food composition containing combined extracts comprising Schisandrae fructus, Eucommiae cortex and Lycii fructus as active ingredients for preventing or treating metabolic bone diseases. The composition of the present invention does not have side effects as it is a substance derived from natural sources; is effective on osteogenesis in ovariectomized mice; increases the bone content and the bone formation index of serum in real animal model; and reduces the bone resorption index so as to be useful for preventing or treating metabolic bone diseases.

Claims

1. A pharmaceutical composition containing combined extracts comprising Schisandrae fructus, Eucommiae cortex and Lycii fructus as active ingredients for preventing or treating metabolic bone diseases.

2. The pharmaceutical composition according to claim 1, wherein a weight ratio of Schisandrae fructus, Eucommiae cortex and Lycii fructus is 1˜100:1˜100:1˜100.

3. The pharmaceutical composition according to claim 2, wherein the extract is extracted with an extraction solvent selected from a group consisting of alcohol, C.sub.1˜C.sub.4 alcohol, and a mixture thereof.

4. The pharmaceutical composition according to claim 1, wherein the metabolic bone diseases are selected from a group consisting of osteoporosis, osteopenia, osteomalacia, osteodystrophy and Paget's disease.

5. The pharmaceutical composition according to claim 4, wherein osteoporosis is postmenopausal osteoporosis.

6. A food composition containing combined extracts comprising Schisandrae fructus, Eucommiae cortex and Lycii fructus as active ingredients for preventing or improving metabolic bone diseases.

7. A method for preventing or treating metabolic bone diseases by administering the pharmaceutical composition according to claim 1 to a subject in need.

8. (canceled)

9. A method for preventing or treating metabolic bone diseases by administering the pharmaceutical composition according to claim 2 to a subject in need.

10. A method for preventing or treating metabolic bone diseases by administering the pharmaceutical composition according to claim 3 to a subject in need.

11. A method for preventing or treating metabolic bone diseases by administering the pharmaceutical composition according to claim 4 to a subject in need.

12. A method for preventing or treating metabolic bone diseases by administering the pharmaceutical composition according to claim 5 to a subject in need.

13. A method for preventing or improving metabolic bone diseases by administering the food composition according to claim 6 to a subject in need.

Description

DESCRIPTION OF DRAWINGS

[0064] FIG. 1 is a photograph confirming changes in growth plate thickness for femurs in each administration group.

[0065] FIG. 2 is a graph comparing changes in a bone mineral content in femurs.

[0066] FIG. 3 is a graph comparing changes in a bone mineral density in femurs.

[0067] FIG. 4 is a result confirming expressions of Runx2 and Osterix in Saos-2 cells.

BEST MODE

[0068] The preferred preparation examples and examples are introduced as follows to help the understanding of the present invention. However, these preparation examples and examples are merely to assist the understanding of the invention, but do not limit the scope of the invention.

[0069] Hereinafter, the present invention will be described in further details with reference to examples, but the scope of the present invention is not limited only to the examples. Further, significances amongst control group, comparison group and test group, as per results of the following experiments were compared multiple times using Scheffe method and Bonferroni method after having been verified by ANOVA at a=0.5 level.

Preparation Example 1. Preparation of the Combined Extracts of the Present Invention

[0070] After having been dried, Schisandrae fructus, Eucommiae cortex, and Lycii fructus purchased from Jeongdosaengyak Co. (Seoul) were measured to what shown in Table 1, and grinded. Then 70% ethanol, which is equivalent of 10 times the weight of the specimen, was added before it was extracted for 24 hours at room temperature. The obtained extract was concentrated under reduced pressure using a rotary vacuum concentrator to obtain concentrates, which would then freeze at −70 □ by a lyophilizer and turn into a powder form. The obtained lyophilized powder is then used as the final extract in the experiments below.

TABLE-US-00001 TABLE 1 Schisandrae Eucommiae fructus cortex Lycii fructus Preparation Example 1 200 g 100 g 100 g

Preparation Examples 2 to 4. Preparing Schisandra fructus Extract, Eucommiae cortex Extract and Lycii fructus Extract

[0071] Except for extracts that were prepared solely on their own according to the weights indicated in Table 2, single extracts of Schisandra fructus, Eucommiae cortex and Lycii fructus were prepared separately in the same way illustrated in Preparation Example 1.

TABLE-US-00002 TABLE 2 Schisandrae Eucommiae fructus cortex Lycii fructus Preparation 400 g Example 1 Preparation 400 g Example 2 Preparation 400 g Example 3

MODE FOR INVENTION

Example 1. Confirming Treatment Effects on Metabolic Bone Diseases in Ovariectomized Mice

1-1. Delivering Ovarian Hysterectomy and Medication

[0072] In order to measure a treatment activity of the combined extracts of Preparation Example 1 on metabolic bone diseases, 28 six-weeks-old white female ICR mice with an average body weight of 30 g (Raon Bio Co., the Republic of Korea) were bred at 22 to 24 □ in temperature and 55 to 60% in relative humidity. The animals were fed with solid feeds (Daehan Biolink Co., the Republic of Korea), and freely accessible to food and water.

[0073] Test animals were divided into {circle around (1)} normal group, where suturing operations on stomachs after confirming ovaries by cutting (sham operations) were performed; {circle around (2)} control group (OVX), where ovariosteresis were performed and no drugs were administered; {circle around (3)} positive control group (E2), where ovariosteresis were performed and estradiol (estradiol; E2), the mammalian estrogen was administered; {circle around (4)} combined group, where 200 mg/kg of the combined extracts of the present invention (Preparation Example 1) were administered; {circle around (5)} Schisandrae fructus group, where 100 mg/kg of the Schisandrae fructus-only extract of the present invention (Preparation Example 2) was administered; {circle around (6)} Eucommiae cortex group, where 50 mg/kg of the Eucommiae cortex-only extract (Preparation Example 3) was administered; and {circle around (7)} Lycii fructus group, where 50 mg/kg of the Lycii fructus-only extract (Preparation Example 4) was administered.

[0074] Ovariosteresis was performed after anesthetizing test animals with a mixture of Zoletil and Rumpun 1:1 (volume ratio). The animals were injected with 0.002 mL of the mixture per 1 g of their body weight. When the animals reached deep anesthesia, ovaries were removed from their incised kidneys that were located at both sides of the spines, and the incision sites were sutured.

[0075] The above combined extracts and single extracts were orally administered daily with an experimental specimen where powders shown in Preparation Examples 1 to 4 at a dosage, where powders were dissolved in 200 μl of distilled water, from the sixth week after the ovariosteresis. Normal group and control group were administered with the same amount of distilled water used in the test group. Positive control group was peritoneally administered daily with dosage thereof, along with 10 μg/kg estradiol dissolved in 200 μl distilled water from the sixth week after the ovariosteresis.

1-2. Measuring Thickness of Growth Plate

[0076] The collected femoral bone tissues were decalcified in an aqueous formic acid fixed with 10% formalin. An observation area of the bone tissues was dissected by a scalpel, dehydrated in a 70% to 100% of alcohol and acetone, and then washed with xylene before carrying out paraffin-embedding. The paraffin-embedded bone tissues were then sectioned to 5 μm with a microhm and stained with hematoxyline and eosin (H&E), of which result was confirmed with an optical microscope (see FIG. 1).

[0077] The thickness of growth plate in the ovariectomized control group (OVX) rapidly surged, compared to the same in normal group, whereas the thickness of femoral growth plate administered with the combined extracts of the present invention was visibly reduced. Therefore, it is proved that the combined extracts of the present invention improves histological symptoms of osteoporosis by suppressing changes in bone tissues.

1-3. Comparing Femoral Bone Mineral Content and Femoral Bone Mineral Density.

[0078] The collected femur was used to compare a femoral bone mineral density. The epiphysis of the left femur was dissected, and treated for a dual energy X-ray absorption analysis using a PIXImus device. The femoral bone mineral content (BMC) and the femoral bone mineral density (BMD) obtained from mice in each control group and test group were then compared (see FIG. 2 and FIG. 3).

[0079] The bone mineral content and bone mineral density in the ovariectomized control group (OVX) were significantly reduced, compared to normal group thereof. Femoral BMC and BMD were recovered to a comparable level found in positive control group when the combined extracts of the present invention was administered. This confirmed superior efficacy of the combined extracts disclosed in the present invention in comparison with the single extracts of Schisandrae fructus, Eucommiae cortex and Lycii fructus, respectively. Hence, the combined extracts of the present invention may be useful to prevent or treat metabolic bone diseases such as osteoporosis by suppressing BMC and BMD.

Example 2. Confirming Expressions of Runx2 and Osterix in Saos-2 Cells

[0080] To confirm expressions of factors associated with osteoblast proliferation, test substances were treated for 24 hours in Saos 2 cells, which are osteoblast-like cell line.

[0081] The concentration of the test substance was treated with 100 μg/mL of the combined extract, 50 μg/mL of Schisandrae fructus extract, 25 μg/mL of Lycii fructus extract, and 25 μg/mL of Eucommiae cortex extract. The expression of Osterix and Runx2 (Runt-related transcription factor 2), which is an osteoblast proliferation accelerating factor, was then confirmed by a Western blot assay.

[0082] As a result, the combined extracts of the present invention significantly increased the expression of factors associated with osteoblastic proliferation, of which level was remarkably greater than the same in the single administration groups of Schisandrae fructus, Eucommiae cortex and Lycii fructus, respectively.

<Formulation Example> Preparation of the Pharmaceutical Compositions Comprising the Combined Extracts of the Present Invention

A. Preparing Powder Formulation

[0083] Combined extracts 200 mg

Lactose 100 mg

Talc 10 mg

[0084] Mix the above components and fill them in an airtight pack to produce powders.

B. Preparing Tablet Formulation

[0085] Combined extracts 200 mg
Corn starch 100 mg

Lactose 100 mg

[0086] Magnesium stearate 2 mg
Mix the above components and produce tablets by tableting, according to a conventional tablet preparation method.

C. Preparing Capsule Formulation

[0087] Combined extracts 200 mg
Crystalline cellulose 3 mg

Lactose 14.8 mg

[0088] Magnesium stearate 0.2 mg
Mix the above components and fill them in a gelatin capsule, according to a conventional capsule preparation method.

D. Preparing Injection Formulation

[0089] Combined extracts 200 mg

Mannitol 180 mg

[0090] Injectable sterile distilled water 2974 mg
Na.sub.2HPO.sub.4 12H.sub.2O 26 mg
Prepare each ampule (2 ml) with the above contents, according to a conventional preparation method for injection.

E. Preparing Liquid Formulation

[0091] Combined extracts 200 mg

Isomerose 10 g

Mannitol 5 g

[0092] Lemon flavouring Appropriate amount
Purified water Appropriate amount
Dissolve the above components in purified water, according to a conventional method for preparing liquid formulation and add an appropriate amount of lemon flavours. Then, blend all and adjust amount thereof with purified water to a total of 100 ml before filling it in a brown bottle and sterilizing thereof.
Simple modifications or changes of the present invention may be easily implemented by a person of ordinary skill in the art, thus such modifications or changes are considered to be included within the scope of the invention.

INDUSTRIAL APPLICABILITY OF THE PRESENT INVENTION

[0093] The combined extracts of the present invention may be useful for the prevention or treatment of metabolic bone diseases.