COMPOSITION FOR PREVENTING OR TREATING METABOLIC BONE DISEASES OR MENOPAUSAL SYMPTOMS

Abstract

The present invention relates to a composition for preventing or alleviating metabolic bone diseases or menopausal symptoms, comprising a complex extract of Pueraria radix and Platycodon grandiflorum as an active ingredient. When the present invention is used, metabolic bone disease or menopausal symptoms can be effectively prevented or alleviated without side effects.

Claims

1. A pharmaceutical composition for preventing or treating a metabolic bone disease or a menopausal symptom, the composition comprising: (a) a complex extract of Pueraria lobata and Platycodon grandiflorum as an active ingredient; and (b) a pharmaceutically acceptable carrier thereof.

2. The composition of claim 1, wherein a weight ratio (w/w) of Pueraria lobata to Platycodon grandiflorum in the complex extract is 1:20 to 20:1.

3. The composition of claim 1, wherein the extract is a polar organic solvent extract.

4. The composition of claim 3, wherein the polar organic solvent is water, an anhydrous or hydrated lower alcohol of 1-4 carbon atoms, acetic acid, or a mixture thereof.

5. A food composition for preventing or alleviating a metabolic bone disease or a menopausal symptom, the composition comprising a complex extract of Pueraria lobata and Platycodon grandiflorum as an active ingredient.

6. The food composition of claim 5, wherein a weight ratio (w/w) of Pueraria lobata to Platycodon grandiflorum in the complex extract is 1:20 to 20:1.

7. The food composition of claim 5, wherein the extract is a polar organic solvent extract.

8. The food composition of claim 7, wherein the polar organic solvent is water, an anhydrous or hydrated lower alcohol of 1-4 carbon atoms, acetic acid, or a mixture thereof.

9. A method for preventing, alleviating, or treating metabolic bone diseases or menopausal symptoms, the method comprising: administering to a subject in need thereof, a pharmaceutical composition or food composition comprising the complex extract of Pueraria lobata and Platycodon grandiflorum.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0066] FIG. 1 shows a TRAP activity reduction effect of a complex extract of Pueraria lobata and Platycodon grandiflorum on differentiated osteoclasts according to Experimental Example 1. ###P<0.001 vs. Pueraria lobata group; *** P<0.001 vs. Platycodon grandiflorum group (one-way ANOVA, with Bonferroni's multiple comparison test).

[0067] FIG. 2 shows a TRAP activity reduction effect of a complex extract for each of hot-water and ethanol concentrations on differentiated osteoclasts according to Experimental Example 1. *** P<0.001 vs. negative control group (one-way ANOVA, with Bonferroni's multiple comparison test).

[0068] FIG. 3 shows a vasorelaxation effect of a complex extract of Pueraria lobata and Platycodon grandiflorum on a thoracic aorta section according to Experimental Example 2. ###P<0.001 vs. Pueraria lobata group; * P<0.05 vs. Platycodon grandiflorum group (one-way ANOVA, with Bonferroni's multiple comparison test).

[0069] FIGS. 4, 5, 6 and 7 show an effect of a complex extract of Pueraria lobata and Platycodon grandiflorum on blood ALP (FIG. 4), Ca (FIG. 5), MMP-9 (FIG. 6), and osteocalcin (FIG. 7) concentrations compared with a ovariectomy group (OVX+Vehicle) according to Experimental Example 3. * P<0.05 vs. negative control group (OVX+Vehicle) (one-way ANOVA, with Dunnett's multiple comparison test).

DETAILED DESCRIPTION

[0070] Hereinafter, the present disclosure will be explained in detail with reference to examples. These examples are provided only for the purpose of illustrating the present disclosure in more detail, and therefore, according to the purpose of the present disclosure, it would be apparent to a person skilled in the art that these examples are not construed to limit the scope of the present disclosure.

EXAMPLES

Preparation Example 1: Preparation of Complex Extract of Pueraria lobata and Platycodon grandiflorum

[0071] Washed and dried Pueraria lobata and Platycodon grandiflorum were mixed at a weight ratio (w/w) of 1:1, 2:1, 4:1, 8:1, 1:2, 1:4, and 1:8 and added with 10 volumes 70% aqueous ethanol, followed by extraction at 20° C. for 72 hours while stirring well. Thereafter, filtration was conducted before vacuum concentration at 45-50° C. Lyophilization afforded a complex extract powder. The yield is given in Table 1 below.

TABLE-US-00001 TABLE 1 Classification Type of medicinal herb Yield (%) Preparation Example 1-1 Pueraria lobata • Platycodon 21.82 grandiflorum (1:1) Preparation Example 1-2 Pueraria lobata • Platycodon 18.99 grandiflorum (1:2) Preparation Example 1-3 Pueraria lobata • Platycodon 20.08 grandiflorum (1:4) Preparation Example 1-4 Pueraria lobata • Platycodon 19.52 grandiflorum (1:8) Preparation Example 1-5 Pueraria lobata • Platycodon 23.48 grandiflorum (2:1) Preparation Example 1-6 Pueraria lobata • Platycodon 22.12 grandiflorum (4:1) Preparation Example 1-7 Pueraria lobata • Platycodon 22.53 grandiflorum (8:1)

Comparative Example 1: Preparation of Single Extract

[0072] Washed and dried Pueraria lobata and Platycodon grandiflorum were added with 10 volumes 70% ethanol aqueous solution to each medicinal herb weight, followed by extraction 20° C. for 72 hours while stirring well. Thereafter, filtration was conducted before vacuum decompression concentration at 45-50° C. Lyophilization afforded a total of two kinds of single extract powders. The yield is given in Table 2 below.

TABLE-US-00002 TABLE 2 Classification Type of medicinal herb Yield (%) Comparative example 1-1 Pueraria lobata 21.79 Comparative example 1-2 Platycodon grandiflorum 21.56

Preparation Example 2: Preparation of Complex Extract of Pueraria lobata and Platycodon grandiflorum Using Various Extract Solvents

[0073] Washed and dried Pueraria lobata and Platycodon grandiflorum were mixed at a weight ratio (w/w) of 1:1. For a hot water extract, the mixture was subjected to extraction under reflux at 90±5° C. for 3 hours. For an ethanol extract, the mixture was added with 10 volumes of 0, 25, 50, 75, 95% aqueous ethanol before extraction at a 20° C. for 72 hours while stirring well. Then, concentration in a vacuum at 45-50° C. was followed by lyophilization to give a complex extract powder. The yield is given in Table 3 below.

TABLE-US-00003 TABLE 3 Classification Type of medicinal herb Extract solvent Yield (%) Preparation Pueraria lobata • Platycodon Distilled water (hot water) 28.31 Example 1-8 grandiflorum (1:1) Preparation Pueraria lobata • Platycodon  0% Ethanol (cold extraction) 24.68 Example 1-9 grandiflorum (1:1) Preparation Pueraria lobata • Platycodon 25% Ethanol (cold extraction) 27.72 Example 1-10 grandiflorum (1:1) Preparation Pueraria lobata • Platycodon 50% Ethanol (cold extraction) 25.20 Example 1-11 grandiflorum (1:1) Preparation Pueraria lobata • Platycodon 75% Ethanol (cold extraction) 15.41 Example 1-12 grandiflorum (1:1) Preparation Pueraria lobata • Platycodon 95% Ethanol (cold extraction) 2.996 Example 1-13 grandiflorum (1:1)

Experimental Example 1: Inhibitory Effect of Complex Extract on RANKL (Receptor Activator of NF-Kappa B Ligand, Sigma-Aldrich, US)-Induced Osteoclast Differentiation

[0074] TRAP (tartrate-resistant acid phosphatase) is an enzyme used as an indicator of the osteoclast differentiation process, and the reduction in TRAP activity accounts for the inhibition of osteoclast differentiation. In this experiment, osteoclasts which were derived and differentiated by RANKL (receptor activator of NF-kappa B ligand, Sigma-Aldrich, US) from the mouse macrophage cell line RAW264.7 were used to identify effects of the single and complex extracts on osteoclast differentiation through measurement of TRAP activity, and effects of complex extracts on osteoclast differentiation for each solvent.

[0075] 1-1. Cell Line Culture

[0076] First, RAW264.7 cells were incubated using a phenol-red free α-MEM (GIBCO, USA) medium supplemented with 10% charcoal-stripped fetal bovine serum (GIBCO) in a 5% CO2 incubator at 37° C. RAW264.7 cells were plated to a 96-well plate at a density of 1×10.sup.3 cells per well, and stabilized for 24 hours. Thereafter, the cells were treated with 50 ng/ml of RANKL and 600 μg/ml of each of single and complex extracts of Pueraria lobata and Platycodon grandiflorum or 600 μg/ml of each of a hot water complex extract and ethanol complex extracts of Pueraria lobata and Platycodon grandiflorum for 72 hours before TRAP activity analysis.

[0077] 1-2. TRAP Activity

[0078] In brief, the cells were incubated with 30 μl of TRAP activity assay solution (sodium acetate 600 mM, pH 5.5, L-ascorbic acid 17.6 mg/ml, sodium-tartrate dehydrate 9.2 mg/ml, 4-nitrophenylphosphate Na 3.6 mg/ml, Triton X-100 0.3%, EDTA 6 mM, NaCl 600 mM) for 30 minutes in 37° C. incubator. After the reaction was terminated with 300 mM NaOH absorbance was read at 450 nm wavelength.

[0079] TRAP activity is expressed as a ratio relative to the RANKL-treated group (negative control).

[0080] In the experiment for comparison between the single extracts and the complex extracts, treatment of RAW264.7 with RANKL increased TRAP activity, compared the untreated group, which was reduced in all of the extract-treated groups, as shown in Table 4 and FIG. 1. Particularly, the complex extract-treated groups further decreased in TRAP activity compared to groups treated with single extracts of Pueraria lobata or Platycodon grandiflorum, demonstrating that the complex extracts highly effectively inhibit osteoclast differentiation, compared with the single extracts.

TABLE-US-00004 TABLE 4 TRAP Experimental Pueraria lobata:Platycodon activity Group grandiflorum ratio (%) Untreated — 14.24 ± 1.49 RANKL-treated — .sup. 100 ± 7.03 Pueraria lobata — 22.59 ± 2.21 extract (comparative example 1-1) Platycodon grandiflorum — 22.98 ± 1.04 extract (comparative example 1-2) Complex extract 1:1  7.76 ± 0.27 (preparation example 1-1) Complex extract 1:2  6.21 ± 0.21 (preparation example 1-2) Complex extract 1:4  6.29 ± 0.07 (preparation example 1-3) Complex extract 1:8  6.02 ± 0.05 (preparation example 1-4) Complex extract 2:1 29.57 ± 0.64 (preparation example 1-5) Complex extract 4:1 30.99 ± 4.75 (preparation example 1-6) Complex extract 8:1 37.16 ± 4.87 (preparation example 1-7)

[0081] As shown in following Table 5 and FIG. 2, remarkable TRAP activity reduction was found in all experimental groups treated with the hot-water extract (preparation example 1-8), and the cold-ethanol extracts according to concentrations (preparation examples 1-9, 1-10, 1-11, 1-12, and 1-13).

TABLE-US-00005 TABLE 5 TRAP Experimental Pueraria lobata:Platycodon activity Group grandiflorum ratio (%) Untreated — 23.75 ± 2.42 RANKL-treated —   100 ± 14.72 Hot water extract 1:1 27.76 ± 1.98 (preparation example 1-8)  0% Cold ethanol 1:1 19.74 ± 1.74 complex extract (preparation example 1-9) 25% Cold ethanol 1:1 20.89 ± 1.90 complex extract (preparation example 1-10) 50% Cold ethanol 1:1 19.52 ± 1.78 complex extract (preparation example 1-11) 75% Cold ethanol 1:1 18.04 ± 0.68 complex extract (preparation example 1-12) 95% Cold ethanol 1:1 18.18 ± 0.95 complex extract (preparation example 1-13)

[0082] In addition, correction of the values in Tables 4 and 5 confirmed similar TRAP activity between Preparation Example 1-1 in Table 4 and Preparation Example 1-12 of Table 5.

Experimental Example 2: Vasorelaxation Efficacy of Pueraria lobata and Platycodon grandiflorum Complex Extract

[0083] The estrogen deficiency is a cause of blood pressure elevation in menopausal women by inducing vasoconstriction. Cerebrovascular constriction induces cerebral apoplexy. Therefore, a vasorelaxation efficacy is used as an important evaluation indicator in finding candidate materials of prophylactic and therapeutic drugs for menopausal cardiovascular diseases. In order to identify vasorelaxation efficacy of the complex extracts of Pueraria lobata and Platycodon grandiflorum of Preparation Example 1, phenylephrine (PE)-precontracted rat thoracic aortic strips were assayed for relaxation response in an organ bath.

[0084] 2-1. Preparation of Thoracic Aortic Strip

[0085] Thoracotomy was performed on the experimental animals male Sprague Dawley (SD) rats at 8 weeks of age that were anesthetized with ethylether inhalation. Immediately after being excised, thethoracic aorta was supplied with a gas of 95% of 02 and 5% of CO2 and cleaned of adherent tissues and fats in Krebs-Henseleit solution (K—H solution, composition, mM: NaCl, 118.0; KCl, 4.7; MgSO.sub.4, 1.2; KH.sub.2PO.sub.4, 1.2; CaCl.sub.2, 2.5; NaHCO.sub.3, 25.0; and glucose, 11.1; pH 7.4) maintained at 37° C. Subsequently, the aorta was cut into strips in a ring form about 2 mm in length.

[0086] 2-2. Isotonic Contraction

[0087] The prepared aortic strip was hooked at the opposite ends thereof by respective tungsten wires, after which the lower portion thereof was anchored to a hook installed on the bottom of a 10-mL organ bath while the upper portion thereof was connected to an isometric force transducer (AD Instrument Co., Australia) connected to a physiograph (AD Instrument Co., Australia) for the measurement of isotonic contraction changes, which was recorded using the PowerLab program (AD Instrument Co., Australia). The strip was stabilized for 15 minutes in the organ bath and then loaded with a passive tension of 1 g. An experiment was proceeded after re-stabilization for 1 hour. The Krebs-Henseleit solution in the organ bath was replaced with a fresh one every 20 minutes during stabilization.

[0088] In order to identify a vasorelaxation activity therein, the aortic strip was treated with 1 μM phenylephrine for 40 minutes in the organ bath to induce vasocontraction. The sufficiently contracted strip was treated with 1 mg/ml of each of the single and complex extracts of Pueraria lobata and Platycodon grandiflorum in Krebs-Henseleit solution in the organ bath to compare vasorelaxation activities. The negative control was treated with Krebs-Henseleit solution alone. The vasorelaxation rate (%) was calculated by the following equation.

[00001]$\begin{array}{cc}\mathrm{vasorelaxation}\mathrm{rate}\left(\%\right)=\frac{\left(B-A\right)-\left(C-A\right)}{\left(B-A\right)}\times 100& \mathrm{Equation}\end{array}$

[0089] A: strip contraction before PE-induced contraction

[0090] B: strip contraction after PE-induced contraction

[0091] C: strip contraction after K—H solution or extract treatment

[0092] As shown in Table 6 and FIG. 3, treatment of the aortic strips with phenylephrine induced vasorelaxation at a rate of about 19.92%. Compared with the negative control, the single extract of Pueraria lobata exhibited no effects on vasorelaxation whereas treatment with the Platycodon grandiflorum extract and the complex extract increased the vasorelaxation rate by about 31.35% and about 46.26%, respectively, thus demonstrating the complex extract according to the present disclosure was superior to the negative control and the single extracts in terms of vasorelaxation efficacy.

TABLE-US-00006 TABLE 6 Strip contraction value (g) Before PE- After PE- Experimental induced induced After K-H solution Vasorelaxation Group contraction contraction or extract treatment rate (%) Negative 1.13 ± 0.02 3.38 ± 0.11  2.94 ± 0.1272 19.92 ± 2.70 control Pueraria lobata 1.24 ± 0.02 3.51 ± 0.19 3.11 ± 0.20 18.60 ± 3.42 extract (comparative example 1-1) Platycodon 1.20 ± 0.03 3.25 ± 0.09 2.21 ± 0.13 51.27 ± 4.37 grandiflorum extract (comparative example 1-2) Complex 1.17 ± 0.04 3.57 ± 0.25 1.99 ± 0.17 66.18 ± 4.77 extract (preparation example 1-1)

Experimental Example 3: Efficacy of Complex Extract of Pueraria lobata and Platycodon grandiflorum on Bone Loss Marker

[0093] Estrogen deficiency, which is characteristically observed in menopausal women, induces differentiation of osteoclasts degrading bones, resulting in bone loss and osteoporosis. The ovariectomized (OVX) rat model is currently the best model to reflect estrogen deficiency-induced bone loss symptoms in menopausal women. An ovariectomized rat model was observed in have lowered blood concentrations of calcium (Ca) and inorganic phosphate, which are constituents of bones, and increased levels of MMP-9 (Matrix metallopeptidase-9), which is a main enzymes for degrading bones, ALP (alkaline phosphatase), which is a bone turnover marker, and osteocalcin. These markers are used as main indices for evaluating bone loss. In order to examine an alleviative effect of the complex extracts of Pueraria lobata and Platycodon grandiflorum of Preparation Example 1 on bone loss in terms of the markers, biochemical analysis was conducted on the blood of the ovariectomized female rat.

[0094] 3-1. Construction of Ovariectomized (OVX) Rat

[0095] Female Sprague Dawley rats at 6 weeks of age (each weighing about 160 g±20%) were purchased from Orient (OrientBio, Gapyeong-gun, Gyeonggi-do) and acclimated for 14 days before experiments. The animals were anesthetized with Zoletil 50 (VIRBAC, France) and xylazine (Rompun®, Bayer AG, Germany) for ovariectomy. After the animals were shaved at both the abdominal and dorsal sides and disinfected at sites for surgery, incisions were made in the muscle and peritoneum to expose the uterus and ovaries to identify ovary loci. After exposure of the fallopian tubes and ovarian arteriovenous veins, the ovaries were excised using a cautery, followed by closing the wounds. The test substances, including single and complex extracts of Pueraria lobata (Comparative Example 1-1), Platycodon grandiflorum (Comparative Example 1-2), and Pueraria lobata and Platycodon grandiflorum (Preparation Example 1-1) were orally administrated once a day at a daily dose of 200 mg/kg for 7 days/week, 12 weeks. The normal group and the negative control were orally administered distilled water.

[0096] 3-2. Blood Biochemical Test

[0097] After 12 weeks of administration, a serum separated from blood collected from the posterior vena cava was analyzed for alkaline phosphatase (ALP) and calcium, using a blood biochemical analyzer (7180 Hitachi, Japan) and an automatic electrolyte analyzer. In addition, the osteolysis markers MMP (Matrix metallopeptidase)-9 and Osteocalcin were measured by ELISA.

[0098] As shown in Table 7 and FIGS. 4 to 7, ovariectomy resulted in increasing blood levels of the osteoblast markers ALP and osteocalcin, which increase upon bone loss, and MMP-9, which is directly involved in the breakdown of bones, and decreasing blood levels of Ca, which is an inorganic constituent of bones. When the test substances were administered, there were no significant changes of ALP and Ca levels in the single extract-administered groups, but the markers were changed in a desired direction the complex extract-administered group with significance. In addition, the complex extract-administered group showed decreased blood levels of MMP-9 and osteocalcin markers. From the above results, it was confirmed that the complex extract of the present disclosure has excellent efficacy in improving indicators related to bone loss compared to the negative control and single extracts.

TABLE-US-00007 TABLE 7 Experimental ovariectomy Experimental ALP Ca MMP-9 Osteocalcin Group (OVX) material (U/L) (mg/dL) (ng/mL) (ng/mL) Normal X Distilled water 782.01 ± 89.86 10.02 ± 0.15  7.65 ± 0.61 260.85 ± 23.94 Negative O Distilled water 825.61 ± 76.23 9.74 ± 0.16 9.81 ± 0.73 332.96 ± 37.91 control Pueraria lobata O Comparative 860.26 ± 49.10 9.93 ± 0.12 10.76 ± 1.79  305.26 ± 49.03 extract example 1-1 (comparative example 1-1) Platycodon O Comparative 841.26 ± 56.21 9.89 ± 0.14 12.38 ± 1.86  299.93 ± 22.68 grandiflorum example 1-2 extract (comparative example 1-2) Complex O Preparation 649.19 ± 43.50 10.30 ± 0.14  7.91 ± 1.00 239.89 ± 24.53 extract example 1-1 (preparation example 1-1)